Increased Reporter Gene Expression Research Articles

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

NF-κB subunit RELA suppression of mucosa-associated lymphoid tissue lymphoma translocation protein 1 expression in oral carcinoma cells

Loss of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) expression closely associates with increased aggressive behaviors of oral carcinoma cells. It emphasizes that a mechanism to suppress the expression is an important subject for understanding carcinoma progression pathway. However, nothing is known at present. This study conducted on transcriptional regulation of the gene down-regulation. Reporter assays showed the presence of the silencer region between +402 and +501 region of MALT1 gene in oral carcinoma cells. It encoded a binding site of nuclear factor-κB subunit, RELA. RELA binding to the site was confirmed by the chromatin immunoprecipitation analyses, and deletion and mutations of the site significantly decreased the RELA binding. Short interfering RNAs for RELA up-regulated reporter gene and endogenous MALT1 protein expressions, and deletion and mutations of RELA binding site increased reporter gene expression. These results demonstrated RELA-binding to the site suppresses MALT1 expression that may facilitate oral carcinoma progression.

Exosome-associated SUMOylation mutant AAV demonstrates improved ocular gene transfer efficiency in vivo

Exosome associated Adeno-associated virus (AAV) vectors have emerged as a promising tool in gene therapy. Recently, we elucidated the role of SUMOylation post-translational modification in AAV2 capsid and demonstrated that capsid modifications at SUMOylation sites, enhance vector transduction. The present study was designed to study the combinatorial effect of exosome delivery of a SUMOylation site modified AAV2, during ocular gene therapy. In the first set of experiments, we investigated the in vitro gene transfer potential of exosome-associated SUMOylation mutant AAV2 (Exo-K105Q-EGFP) in human retinal pigmental epithelial (ARPE19) cells. Our data showed that, Exo-K105Q vectors had a significantly higher transduction potential in ARPE19 cells when compared to exosomes derived from wildtype AAV2 (Exo-AAV2-EGFP) vector packaging. Subsequently, an intravitreal administration of exosome associated mutant AAV2 vectors in C57BL6/J mice, demonstrated a significant increase reporter gene (EFGP) expression 4 weeks after gene transfer. Further immunostaining, revealed that these exosome-based vectors also had a better permeation across the retinal layers. These data highlight the translational potential of exosome associated SUMOylation mutant AAV for ocular gene therapy.

Multi-functional self-assembled nanoparticles for pVEGF-shRNA loading and anti-tumor targeted therapy.

Although RNA interference (RNAi) technology shows great potential in cancer treatment, the tumor target delivery and sufficient cytosolic transport of RNAi agents are still the main obstacles for its clinical applications. Herein, we report a functional supramolecular self-assembled nanoparticle vector for RNAi agent loading and tumor target therapy. Molecular block adamantane-grafted poly(ethylene glycol) (Ad-PEG) was modified with epidermal growth factor receptor (EGFR)-specific binding ligand GE11 or pH-sensitive fusogenic peptide GALA and then used for self-assembly with cyclodextrin-grafted branched polyethylenimine (CD-PEI), adamantane-grafted polyamidoamine dendrimer (Ad-PAMAM), and plasmid DNA containing a small hairpin RNA expression cassette against vascular endothelial growth factor (VEGF) into functional DNA-loaded supramolecular nanoparticles (GE11&GALA-pshVEGF@SNPs) based on molecular recognition and charge interaction. These functional peptides facilitated the target cell binding, internalization, and endosomal escape of GE11&GALA-pshVEGF@SNPs, resulting in increased reporter gene expression and efficient targeted gene silencing. The systemic delivery of the GE11&GALA-pshVEGF@SNPs can efficiently downregulate the intratumoral VEGF protein levels, reduce blood vessel formation, and significantly inhibit A549 xenograft tumor growth. These results reveal the potential of these multifunctional self-assembled nanoparticles as a nucleic acid drug delivery system for the treatment of lung cancer.

Carnosine inhibits glioblastoma growth independent from PI3K/Akt/mTOR signaling.

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine’s anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.

Cholesterol regulates cardiac KATP subunit expression through an SREBP‐dependent mechanism

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases the expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP‐sensitive K+ (KATP) channel in HL‐1 cardiomyocytes. Importantly, the effects of TSA were abolished if cells were treated with exogenous cholesterol, suggesting that cholesterol may suppress channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. As expected, treatment of HL‐1 cardiomyocytes with TSA (30 ng/mL) caused a time dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. This is specific for SUR2 and we observe little or no change in expression of the SUR1 subunit. Consistent with a central role for cholesterol‐dependent activation of SREBP in driving the increase in SUR2 mRNA expression, we observe a marked increase in the cleavage of SREBP1 following TSA, and this is inhibited by addition of cholesterol to the media. Further supporting the role of SREBP following TSA treatment, we observe increased expression of other SREBP‐dependent genes including PCSK9 and inducible degrader of LDLr (IDOL). Lastly, in luciferase promoter assays SREBP‐1a markedly increased reporter gene expression driven by the upstream SUR2, but not SUR1, promoter. Taken together, our data are consistent with the conclusion that cholesterol depletion by TSA leads to activation the SREBP signaling pathway that directly acts on the SUR2 promoter to increase gene expression. This finding suggests a novel means by which hypercholesterolemia may exert negative effects on the cardiovascular system by suppressing expression of the KATP channel.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of a Human-Specific Tandem Repeat Associated with Bipolar Disorder and Schizophrenia

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations.

Near-infrared light-controlled systems for gene transcription regulation, protein targeting and spectral multiplexing.

Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.

Modular organization of a hypocretin gene minimal promoter.

Orexins or hypocretins are neurotransmitters produced by a small population of neurons in the lateral hypothalamus. This family of peptides modulates sleep-wake cycle, arousal and feeding behaviors; however, the mechanisms regulating their expression remain to be fully elucidated. There is an interest in defining the key molecular elements in orexin regulation, as these may serve to identify targets for generating novel therapies for sleep disorders, obesity and addiction. Our previous studies showed that the expression of orexin was decreased in mice carrying null-mutations of the transcription factor early B-cell factor 2 (ebf2) and that the promoter region of the prepro-orexin (Hcrt) gene contained two putative ebf-binding sites, termed olf-1 sites. In the present study, a minimal promoter region of the murine Hcrt gene was identified, which was able to drive the expression of a luciferase reporter gene in the human 293 cell line. Deletion of the olf1-site proximal to the transcription start site of the Hcrt gene increased reporter gene expression, whereas deletion of the distal olf1-like site decreased its expression. The lentiviral transduction of murine transcription factor ebf2 cDNA into 293 cells increased the gene expression driven by this minimal Hcrt-gene promoter and an electrophoretic mobility shift assays demonstrated that the distal olf1-like sequence was a binding site for ebf2.

The nutrigenetic influence of the interaction between dietary vitamin E and TXN and COMT gene polymorphisms on waist circumference: a case control study.

BackgroundAbdominal obesity (AO) is a common modifiable risk factor for certain non-communicable diseases associated with enhanced oxidative stress (OS). The objective of this work was to investigate whether the interaction between antioxidant vitamin intake and OS-related polymorphisms modulates gene-associated anthropometry in a Spanish population.MethodsA total of 246 subjects with AO, and 492 age and gender matched non-AO subjects were included in the study. Anthropometric, biochemical, and OS parameters, and antioxidant dietary intake data were assessed using validated procedures. DNA from white blood cells was isolated and the genotype of seven polymorphisms from genes involved in OS (pro-oxidant and antioxidant) were analyzed using the SNPlex system. The effects of the c.-793T > C polymorphism on promoter activity and thus thioredoxin (TXN) activity were examined using reporter assays.ResultsThe AO group had higher 8-Oxo-2′-deoxyguanosine levels and took in less vitamin A and vitamin E compared to the non-AO group. Logistic regression analysis revealed that the rs2301241 polymorphism in TXN and rs740603 in catechol-O-methyltransferase (COMT) were associated with waist circumference (WC) and AO. Moreover, these polymorphisms were more strongly associated with variations in WC in subjects with low vitamin E intakes. A promoter assay revealed that the T to C conversion at c.-793 (rs2301241) induced a more than two fold increase in reporter gene expression.ConclusionsWC is associated both with dietary vitamin E intake and genetic variants of TXN and COMT suggesting that existence of a complex nutrigenetic pathway that involves regulation of AO.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0652-4) contains supplementary material, which is available to authorized users.

Tailoring the Surface of a Gene Delivery Vector with Carboxymethylated Dextran: A Systematic Analysis.

Polymeric nanocarriers are attractive nonviral vectors for gene delivery purposes in vivo. For such applications, numerous physiological and subcellular bottlenecks have to be overcome. In that endeavor, each structural feature of nanocarriers can be optimized with respect to its corresponding challenges. Here, we focused on the interface between a model gene delivery nanocarrier and relevant constituents of the physiological environment. We screened a library of carboxymethylated dextrans (CMD) for the electrostatic coating of positively charged nanocarriers. We evaluated the jointed influence of the CMD molecular weight and charge density upon nanocarrier coating with respect to DNase, small ions, plasma proteins, red blood cells, and target cells. A total of 4 out of 26 CMD coated nanocarriers successfully passed every screening assay, but did not yield increased reporter gene expression in target cells compared to uncoated nanocarriers. The fine-tuning of CMD for nanocarrier coating yielded a relevant shortlist of candidates that will be further tested in vivo.

Design criteria for synthetic riboswitches acting on transcription

Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

Regulation of the JUB1 stress-related transcription factor in Arabidopsis thaliana in response to biotrophic fungus Oidium neolycopersici

Biotic stresses influence fitness of the plants and may decrease their productivity. Especially biotrophic fungi govern complex regulation of host metabolism to fulfill their nutritional needs, while the plant tries to avoid this fungal activity. The defense system of the plant may involve transcription of stress-dependent genes, activation of signaling pathways or production of antimicrobial compounds. The responsible signal molecule in biotrophic pathogen-host interaction was found to be mostly the salicylic acid (SA). We studied here, whether the salicylic acid (SA) is required for the induction of a stress-related transcription factor JUB1 by the biotrophic fungus of tomato Oidium neolycopersici or not. To prove the regulation of JUB1, we isolated the upstream region of the gene and transcriptionally fused to gus reporter gene. This promoter::reporter fusion was then transferred into wild type and two salicylic acid insensitive mutants of Arabidopsis thaliana (nim1-1 and nahG). The transgenic plants were infected by powdery mildew (PM) O. neolycopersici. We found an obvious increase of reporter gene expression in all three lines mostly at the area, where the pathogen was contacted to the plants. Based on the result, the stress-dependent JUB1 transcription factor is probably not influenced by the SA mutations of nim1-1 and nahG. However, the induction appeared along with hydrogen-peroxide development, which suggests that this gene regulation in response to powdery mildew is probably regulated via H2O2 and not by SA signaling.

Genetic Variants in CCNB1 Associated With Differential Gene Transcription and Risk of Coronary In-Stent Restenosis

The development of diagnostic tools to assess restenosis risk after stent deployment may enable the intervention to be tailored to the individual patient, for example, by targeting the use of drug-eluting stent to high-risk patients, with the goal of improving safety and reducing costs. The CCNB1 gene (encoding cyclin B1) positively regulates cell proliferation, a key component of in-stent restenosis. Therefore, we hypothesized that single-nucleotide polymorphisms in CCNB1 may serve as useful tools in risk stratification for in-stent restenosis. We identified 3 single-nucleotide polymorphisms in CCNB1 associated with increased restenosis risk in a cohort of 284 patients undergoing coronary angioplasty and stent placement (rs350099: TT versus CC+TC; odds ratio [OR], 1.82; 95% confidence interval [CI], 1.09-3.03; P=0.023; rs350104: CC versus CT+TT; OR, 1.82; 95% CI, 1.02-3.26; P=0.040; and rs164390: GG versus GT+TT; OR, 2.27; 95% CI, 1.33-3.85; P=0.002). These findings were replicated in another cohort study of 715 patients (rs350099: TT versus CC+TC; OR, 1.88; 95% CI, 0.92-3.81; P=0.080; rs350104: CC versus CT+TT; OR, 2.23; 95% CI, 1.18-4.25; P=0.016; and rs164390: GG versus GT+TT; OR, 1.87; 95% CI, 1.03-3.47; P=0.040). Moreover, the haplotype containing all 3 risk alleles is associated with higher CCNB1 mRNA expression in circulating lymphocytes and increased in-stent restenosis risk (OR, 1.43; 95% CI, 1.00-1.823; P=0.039). The risk variants of rs350099, rs350104, and rs164390 are associated with increased reporter gene expression through binding of transcription factors nuclear factor-Y, activator protein 1, and specificity protein 1, respectively. Allele-dependent transcriptional regulation of CCNB1 associated with rs350099, rs350104, and rs164390 affects the risk of in-stent restenosis. These findings reveal these common genetic variations as attractive diagnostic tools in risk stratification for restenosis.

Synthetic TAL effectors for targeted enhancement of transgene expression in plants

Transcription activator-like effectors (TALEs), secreted by the pathogenic bacteria Xanthomonas, specifically activate expression of targeted genes in plants. Here, we designed synthetic TALEs that bind to the flanking regions of the TATA-box motif on the CaMV 35S promoter for the purpose of understanding the engineerable 'hot-spots' for increasing transgene expression. We demonstrated that transient expression of de novo-engineered TALEs using agroinfiltration could significantly increase reporter gene expression in stable transgenic tobacco expressing the orange fluorescent protein reporter gene pporRFP under the control of synthetic inducible, minimal or full-length 35S promoters. Moreover, the additive effects of a combination of two different synthetic TALEs could significantly enhance the activation effects of TALEs on reporter gene expression more than when each TALE was used individually. We also studied the effects of the C-terminal domain and the activation domain of synthetic TALEs, as well as the best 'hot-spots' on the 35S promoter on targeted transgene activation. Furthermore, TALE activation of the Arabidopsis MYB transcription factor AtPAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) in stable transgenic tobacco gave rise to a dark purple colour on infiltrated leaves when driven by four copies of cis-regulatory elements of pathogenesis-related gene (PR1) with enhancer motifs B and A1 from the 35S promoter. These results provide novel insights into the potential applications of synthetic TALEs for targeted gene activation of transgenes in plants.

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p>0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10−6M for Gen and >10−5M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.

The androgen receptor facilitates inhibition of human dopamine transporter (DAT1) reporter gene expression by HESR1 and HESR2 via the variable number of tandem repeats

A functional genetic polymorphism in the 3′-untranslated region (UTR) within exon 15 of the human DAT gene (DAT1) has been described. This 3′-UTR contains a variable number of tandem repeats (VNTR) 40bp in length; many association studies of psychiatric or developmental disorders with this VNTR have been conducted. We previously demonstrated that HESR1 (the Hairy/enhancer of split related transcriptional factor 1 with YRPW motif) and HESR2 reduced DAT reporter gene expression via this 3′-UTR. VNTR allele-dependent altered reporter gene expression was also observed. In the present study, we wanted to clarify the molecular characterization of HESR1 and HESR2, focusing on its cis-element and co-factor. Deletion of the VNTR domain increased reporter gene expression both with and without transfection of HESRs, suggesting that the VNTR inhibits DAT expression, and is responsive to HESRs. In the presence of transfected androgen receptor (AR), activity of the luciferase reporter with the nine-repeat allele (9r) decreased, while that with the ten-repeat allele (10r), the most frequent in the population, increased significantly. Furthermore, co-expression of HESR1 or HESR2 with AR increased the inhibitory effect of the HESRs. Our data indicate that a functional modification occurs when the HESRs are coupled with AR. This HESR–AR interaction could be the molecular basis of sexual dimorphisms in DAT expression, or other dopamine-related behavioral traits.

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: A comparison between ex vivo and in vivo data

Purpose: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis.Materials and methods: A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression.Results: We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo.Conclusions: However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

MiRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes.

Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3'-untranslated region (3'-UTR) revealed five putative miR-939 binding sites. The hiNOS 3'-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3'-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutagenesis of miR-939 bindings sites at +99 or +112 bp in the hiNOS 3'-UTR increased reporter gene expression. Furthermore, intact miR-939 binding sites at +99 or +112 positions were required for posttranscriptional suppression by miR-939. Cytokine stimulation directly increased miR-939 levels in human HC. Transfection of miR-939 inhibitor (antisense miR-939) enhanced cytokine-induced hiNOS protein and increased NO synthesis in vitro in human HC. Finally, cytokine or LPS injection in vivo in mice increased hepatic miR-939 levels. Taken together, these data identify that miR-939 directly regulates hiNOS gene expression by binding in the 3'-UTR to produce a translational blockade. These findings suggest dual regulation of iNOS gene expression where cytokines induce iNOS transcription and also increase miR-939, leading to translational inhibition in a check-and-balance system.