Gene expression in higher eukaryotes is precisely regulated in time and space through the interplay between promoters and gene-distal regulatory regions, known as enhancers. The original definition ...of enhancers implies the ability to activate gene expression remotely, while promoters entail the capability to locally induce gene expression. Despite the conventional distinction between them, promoters and enhancers share many genomic and epigenomic features. One intriguing finding in the gene regulation field comes from the observation that many core promoter regions display enhancer activity. Recent high-throughput reporter assays along with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-related approaches have indicated that this phenomenon is common and might have a strong impact on our global understanding of genome organisation and gene expression regulation.
Promoters and enhancers share architectural and functional properties.
When tested on episomal reporters, many promoters display enhancer activity.
In vivo experiments demonstrated that enhancer like promoters function as bona fide enhancers.
Genetic variants lying in enhancer-like promoters might impact on physiological traits or diseases by altering the expression of distal genes.
Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established ...as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding.
MicroRNAs (miRNAs) are conserved noncoding RNAs that function as posttranscriptional regulators of gene expression. miR-9 is one of the most abundant miRNAs in the brain. Although the function of ...miR-9 has been well characterized in neural progenitors, its role in dendritic and synaptic development remains largely unknown. In order to target miR-9 in vivo, we developed a transgenic miRNA sponge mouse line allowing conditional inactivation of the miR-9 family in a spatio-temporal-controlled manner. Using this novel approach, we found that miR-9 controls dendritic growth and synaptic transmission in vivo. Furthermore, we demonstrate that miR-9-mediated downregulation of the transcriptional repressor REST is essential for proper dendritic growth.
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor ...measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega’s luc2 for reporter and imaging applications.
Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine ...(LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the
(Luc),
, and
transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline PBS) conditioning. At 1 month, bioluminescence was detected
in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined
there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected
in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged
. LacZ expression was predominantly observed in the alveolar regions of the lung.
was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.
Nitrate is a nutrient signal that triggers complex regulation of transcriptional networks to modulate nutrient-dependent growth and development in plants. This includes time- and nitrate ...concentration-dependent regulation of nitrate-related gene expression. However, the underlying mechanisms remain poorly understood. Here we identify NIGT1 transcriptional repressors as negative regulators of the Arabidopsis NRT2.1 nitrate transporter gene, and show antagonistic regulation by NLP primary transcription factors for nitrate signalling and the NLP-NIGT1 transcriptional cascade-mediated repression. This antagonistic regulation provides a resolution to the complexity of nitrate-induced transcriptional regulations. Genome-wide analysis reveals that this mechanism is applicable to NRT2.1 and other genes involved in nitrate assimilation, hormone biosynthesis and transcription. Furthermore, the PHR1 master regulator of the phosphorus-starvation response also directly promotes expression of NIGT1 family genes, leading to reductions in nitrate uptake. NIGT1 repressors thus act in two transcriptional cascades, forming a direct link between phosphorus and nitrogen nutritional regulation.
Stochastic Gene Expression in a Single Cell Elowitz, Michael B.; Levine, Arnold J.; Siggia, Eric D. ...
Science (American Association for the Advancement of Science),
08/2002, Letnik:
297, Številka:
5584
Journal Article
Recenzirano
Clonal populations of cells exhibit substantial phenotypic variation. Such heterogeneity can be essential for many biological processes and is conjectured to arise from stochasticity, or noise, in ...gene expression. We constructed strains of Escherichia coli that enable detection of noise and discrimination between the two mechanisms by which it is generated. Both stochasticity inherent in the biochemical process of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) contribute substantially to overall variation. Transcription rate, regulatory dynamics, and genetic factors control the amplitude of noise. These results establish a quantitative foundation for modeling noise in genetic networks and reveal how low intracellular copy numbers of molecules can fundamentally limit the precision of gene regulation.
ATF6 has two isoforms, ATF6α and ATF6β, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional ...roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6β are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6β activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6β. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6β. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6β.
•We have developed a new cell-based reporter system to monitor activation of ATF6β.•A fusion protein (a synthetic transcription factor domain-ATF6β(aa 350-703)) was expressed as an ER stress sensor.•Regulated intramembrane proteolysis of a fusion protein induces luciferase activity.
Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find ...that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.
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•ceRNA-mediated derepression is typically insensitive to reduced miRNA activity•Extensively paired sites can reduce derepression thresholds by triggering miRNA decay•Weak sites can contribute to target-site competition without imparting repression•Closely spaced sites of the same or different miRNAs cooperatively sequester miRNAs
Denzler et al. show that effects of competing miRNA sites are insensitive to reduced miRNA activity, low-affinity/background miRNA sites contribute to competition, and adjacent miRNA sites can cooperatively sequester miRNAs. Overall, their results reduce the prospects of observing an effect from a ceRNA.
Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) ...system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.
•Recombinant rotaviruses harboring HiBiT tag were generated.•The function of viral NSP1 was not affected by addition of HiBiT tag.•Replication of HiBiT reporter rotaviruses was monitored by NanoLuc activity.•HiBiT reporter rotavirus system is a powerful tool for antiviral screening.
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