Constitutive Gene Activation Research Articles

Autism associated mutations in β2 subunit of voltage-gated calcium channels constitutively activate gene expression

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α11.2, or β2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavβ2a corresponding to autistic related mutations Cavβ2dS197F and Cavβ2dG167S in the human Cavβ2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α11.2 and α2δ with Cavβ2aS143F or Cavβ2aG113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α11.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavβ2a autistic mutants is consistent with Cavβ2/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα11.2/Cavβ2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders.

Constitutive activation of genes involved in triterpene saponins enhances the accumulation of saponins in three-year-old Panax notoginseng growing under moderate light intensity

Light is an important physical factor that regulates plant developmental patterns and induces the synthesis of secondary metabolites. The optimal light intensity for saponin accumulation in roots of medicinal plants with different ages is relatively poorly understood. After that, the correlation of light-driven biosynthesis of triterpene saponins with the expression of genes incorporated into the biosynthesis of triterpene saponins is still unknown. The expression levels of genes, the total saponin contents, and the biomass accumulation and allocation were examined in different age Panax notoginseng grown under different light intensities. A significant elevation in total saponin content was found in three-year-old plants grown under 9.6 % of full sunlight (FL) condition compared with the others. The expression level of dammarenediol-II synthase (DS) rapidly increased from one-year-old plants to three-year-old plants, which was consistent with the change in total saponins. Furthermore, there is a positive relationship between the expression level of DS and the content of saponins. Additionally, 9.6 % FL-driven biosynthesis of saponin has more activity in the three-year-old P. notoginseng, and the genes expression level of CYP716A47 was elevated. At the same time, the expression of geranylgeranyl pyrophosphate synthase (GGPS) and phosphomevalonate kinase (PMK) had no positive contribution to the accumulation of saponins in roots. Overall, DS and CYP716A47 might attribute to the accumulation of saponins in three-year-old P. notoginseng under a moderate light regime (about 10 % FL).

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

Transgenic tobacco plants constitutively expressing peanut BTF3 exhibit increased growth and tolerance to abiotic stresses.

Abiotic stresses limit crop growth and productivity worldwide. Cellular tolerance, an important abiotic stress adaptive trait, involves coordinated activities of multiple proteins linked to signalling cascades, transcriptional regulation and other diverse processes. Basal transcriptional machinery is considered to be critical for maintaining transcription under stressful conditions. From this context, discovery of novel basal transcription regulators from stress adapted crops like peanut would be useful for improving tolerance of sensitive plant types. In this study, we prospected a basal transcription factor, BTF3 from peanut (Arachis hypogaea L) and studied its relevance in stress acclimation by over expression in tobacco. AhBTF3 was induced under PEG-, NaCl-, and methyl viologen-induced stresses in peanut. The constitutive expression of AhBTF3 in tobacco increased plant growth under non stress condition. The transgenic plants exhibited superior phenotype compared to wild type under mannitol- and NaCl-induced stresses at seedling level. The enhanced cellular tolerance of transgenic plants was evidenced by higher cell membrane stability, reactive oxygen species (ROS) scavenging activity, seedling survival and vigour than wild type. The transgenic lines showed better in vitro regeneration capacity on growth media supplemented with NaCl than wild type. Superior phenotype of transgenic plants under osmotic and salinity stresses seems to be due to constitutive activation of genes of multiple pathways linked to growth and stress adaptation. The study demonstrated that AhBTF3 is a positive regulator of growth and stress acclimation and hence can be considered as a potential candidate gene for crop improvement towards stress adaptation.

Updated Phase II Study of Targeted Subcutaneous (SC) Bortezomib for Patients with Low- or Intermediate-1 (Int-1)-Risk Myelodysplastic Syndrome (MDS) with Evidence of NF-κb Activation

▪IntroductionMDS has been linked to constitutive activation of genes involved in the nuclear factor-kappaB (NF-κB) pathway [Wei et al. Leukemia 2013; Braun et al. Blood 2006]. Hence, NF-kB is an attractive therapeutic target in this disease. Bortezomib is a proteosome inhibitor with inhibitory activity against NF-κB. We designed a phase II trial of SC bortezomib in patients with low- to intermediate (low/int-1)-risk MDS and evidence of NF-κB activation to determine its therapeutic activity in these patients.MethodsIn this single-arm phase II study in low/int-1-risk MDS patients, bortezomib was administered at 1.3 mg/m2 SC on days 1, 4, 8, and 11 in a 21-day cycle for a maximum of 2 years of therapy. Eligibility criteria included age ≥18 years, adequate performance status and organ function, and having received at least 1 prior therapy. Patients with grade 2 or greater peripheral neuropathy at baseline were excluded. Patients were prescreened prospectively for enrollment by assessing cellular levels of the phosphorylated NF-κB subunit p65 (pp65) in their marrow. This was performed by immunofluorescence with phospho-Ser276 in bone marrow aspirate smears in the Department of Hematopathology (CLIA regulations). Patients were eligible if at least 5% of all nucleated marrow cells were positive for pp65 staining. pp65 levels were assessed again on day 21 of cycles 1 and 2 and then as clinically indicated. Responses were assessed according to IWG [Cheson et al. Blood 2000; Cheson et al, Blood 2006]. The study could accrue a maximum of 40 patients if there were at least a 95% chance of at least a 15% ORR.ResultsBeginning 9/2013, we enrolled 15 patients with a median age of 71 years (range 56 - 87). Median marrow blast percentage was 1.9% (range 0 - 5%). Eleven patients (73%) had diploid cytogenetics, 2 had del(20q), 1 had Y-, and 1 had del(5q). All had lower-risk MDS by IPSS (low 33.3%, int-1 66.7%). All were transfusion dependent (6 both platelets and red cells (PRBCs), 1 only platelets, 8 only PRBCs). Hypomethylating agents had failed in 12 of the patients.At a median follow-up interval of 22 weeks, the ORR was 20%; 3 patients had hematologic improvement with a mean duration of response of 14.3 weeks (range 4-21). Eight patients had stable disease, and 4 had progression. No correlation between clinical response and molecular or cytogenetic data was observed. Eventually, all patients were taken off study: 7 due to increasing transfusion requirements, 2 worsening cytopenia, 1 lack of response, 1 increased blasts, and 1 grade 2 neuropathy; 2 withdrew by choice, and 1 patient died from causes unrelated to the study. Four patients experienced ≥ 1 grade 3 toxicity. No grade 4 toxicity was observed. Seven patients experienced grade 1 (n=4) or grade 2 (n=3) neuropathy. Morphologic review (n=14) in the responders group (n=3) showed reduction in ring sideroblasts (RS) in 2 patients (60 to 47% and 43% to 30%). Two patients, including 1 of those with reduced RS, had improvement of dysplasia (from severe trilineage dysplasia to moderate bilineage dysplasia). No changes in cytogenetic studies were found in the responder group.Among nonresponders (n=11), 3 had new acquired cytogenetic abnormalities, 3 had worsening dysplasia, and 2 had increase in blast count (1% to 3% and 1% to 4%). Two had no morphologic changes during treatment. One showed improvement in dysplasia and blast count (2% to 0%) and no changes in cytogenetics. Interestingly, 3 nonresponders showed RS reduction during treatment (18% to 1%, 85% to 45%, and 6% to 0%).The median pp65 level at baseline was 30.87% (range 7 - 70%). The pp65 level decreased in 7 of the 15 patients (46.7%), in 6 of them by the end of cycle 1. Interestingly, the 3 responders were among the 7 patients who had a decrease in pp65 level. Eventual loss of response in these patients was accompanied by return to a higher pp65 level. In nonresponders, the pp65 level increased in 7, decreased in 2, and remained unchanged in 1; for 1, the sample was suboptimal.ConclusionsIn previously treated lower-risk MDS patients, SC Bortezomib was well tolerated and resulted in hematologic improvement and decrease in RS. NF-κB activation, measured by pp65 level, can be a useful biomarker to select patients with lower-risk MDS who could benefit from therapies that target this pathway. The NF-κB pattern of expression suggests an inverse relationship between treatment response and NF-κB level, with associated improvement in bone marrow morphology. DisclosuresKonopleva:Calithera: Research Funding; Cellectis: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection.

Dimerization of yeast transcription factors Ino2 and Ino4 is regulated by precursors of phospholipid biosynthesis mediated by Opi1 repressor

Structural genes of phospholipid biosynthesis in the yeast S. cerevisiae are activated by the heterodimeric transcription factor Ino2 + Ino4, binding to ICRE (inositol/choline-responsive element) promoter motifs. In the presence of phospholipid precursors inositol and choline, Ino2-dependent activation is inhibited by the Opi1 repressor which interacts with Ino2. In this work, we systematically investigated the importance of regulatory mechanisms possibly affecting ICRE-dependent gene expression. Autoregulatory expression of INO2, INO4 and OPI1 was abolished by promoter exchange experiments, showing that autoregulation of regulators contributes to the degree of differential gene expression but is not responsible for it. Using GFP fusion proteins, Ino2 and Ino4 were found to localize to the nucleus under conditions of repression and derepression. Interestingly, nuclear localization of Ino2 required a functional INO4 gene. Targeting of a lexA-Ino2 fusion to a heterologous promoter containing lexA operator motifs revealed a constitutive gene activation which was not influenced by phospholipid precursors. We could show that Ino2-dependent activation of a lexA-Ino4 fusion is affected by inositol and choline. Since gene activation required interaction of Ino2 and Ino4 mediated by their helix-loop-helix domains, formation/dissociation of the heterodimer must be considered as an important step of target gene regulation.

Light-Independent Phytochrome Signaling Mediated by Dominant GAF Domain Tyrosine Mutants ofArabidopsisPhytochromes in Transgenic Plants

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.

Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors

Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

Repeated treatment with antidepressants enhances dopamine D 1 receptor gene expression in the rat brain

Many pharmacological investigations have demonstrated that antidepressant agents profoundly affect serotonergic and noradrenergic neurotransmission. The molecular mechanisms by which these drugs exert their therapeutic action have not been clearly established. In our study, the possibility that antidepressant drug action is associated with dopamine neurotransmission was examined. To this end, the effect of 21-day treatment with 10 mg/kg of amitryptyline, mirtazapine and sertraline on the striatal and nucleus accumbens dopamine receptors was verified. The striatum and nucleus accumbens tissues were dissected 24 h after the last dose of the drug and total RNA was isolated. The expression of dopamine D 1 to dopamine D 5 receptors using reverse-transcriptase polymerase chain reaction (RT-PCR) procedure was compared to the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as constitutive gene activation internal control. Lab Works UV program has analyzed the mean optical density values of RT-PCR products. Statistical comparison of relative optical densities by a one-way analysis of variance (ANOVA) followed by Dunnett's test was performed. Despite their different pharmacological profiles, all three above-presented antidepressants significantly increased dopamine D 1 mRNA content. Our findings indicate that repeated antidepressant administration triggers induction of the brain dopaminergic receptors which is correlated with neuroadaptation of the brain dopaminergic pathway.

Effects of active MEK1 expression in vivo

Cell transformation is often a result of constitutive activation of genes in signaling pathways that regulate cell proliferation and differentiation. Indeed, the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway is constitutively activated in a large number of cancers. The extent to which a single-gene mutation can alter cell fate, however, remains questionable. In vitro studies have addressed this issue, but organs are comprised of multiple cell types, and in vitro models often poorly approximate these interactions. In response to these limitations, cell-type specific mouse models have been generated as a means to examine the effect of altering a single element of the MAPK pathway in vivo. This review summarizes data from transgenic murine and human tissue models expressing constitutive active forms of MEK1.

Molecular dynamics of the compensatory response to myocardial infarct

Myocardial infarct via occlusion of the left anterior descending coronary in rats caused overriding depression in transcription, signal transduction, inflammation and extracellular matrix pathways in the infarct zone within 24 h. In contrast, remote zone gene expression was reciprocally activated during the immediate post-infarct period. Infarct zone signal transduction occurred primarily through TGFβ1 induction while the remote zone exhibited elevated WNT, NOTCH, GPCR and transmembrane signaling. A minimal day 1 acute phase, inflammatory response was detected in the infarct zone while interleukins (IL1α, IL1β, IL6, IL12α, IL18) and the TNFα superfamily were activated in the remote zone. Different cytochrome subsets were activated in each left ventricular region on day 1 while anti-oxidant genes were elevated only in the remote zone. The infarct zone exhibited mixed early transcription factor activation across all binding domains with a balance favoring constitutive gene activation and differentiation pathways as opposed to cell proliferation. In contrast, the remote zone exhibited activation of extensive developmental transcription factors involved in specification of cell phenotype, tissue-specific interactions and position-specific cell proliferation on day 1. The day 28 infarct zone response mirrored the day 1 remote zone response including activation of genes associated with matrix remodeling (metallothionein and metalloproteinase 9, 12, 23), as well as genes associated with cell proliferation and phenotype specification (MYC, EGR2, ATF3, HOXA1) recapitulating developmental histogenesis programs.

Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. The PFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA.dT element. Reb1p binds specifically in vitro. Mutation of this site reduces PFY1 expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA.

Influence of the glucocorticoid receptor onc-fos inducibility in activatedras-containing mouse lung cells

Glucocorticoids inhibit the growth and promote the differentiation of normal lung cells. Transformed A5 mouse lung cells containing an activated Ki-ras gene are not responsive to glucocorticoid-induced growth inhibition and demonstrate increased cell proliferation. Activated ras genes may lead to constitutive activation of genes, such as the activating protein 1 (AP-1) transcription factor components fos and jun, which are downstream in the ras signal-transduction pathway. A5 cells and A5GR, a stable A5 transfectant containing excess copies of the glucocorticoid receptor (GR) gene, were examined for potential alterations in AP-1 that accompany the restoration of glucocorticoid-dependent growth inhibition. The established ability of the GR to antagonize AP-1 activity led us to examine the regulation and inducibility of c-fos and c-jun in these cells. Nontransformed C10 lung cells were found to have higher and more inducible AP-1 activity than the transformed A5 cells. The level of AP-1 activity could be reduced in C10 cells by transient transfection of constitutive fos and jun expression vectors. In A5 cells, stimulation with factors that activate the serum-response element on the fos promoter and induce c-fos mRNA had little effect on AP-1 activity, whereas treatment with 12-O-tetradecanoylphorbol-13-acetate, which acts at the fos-AP-1 binding sequence site on the fos promoter, efficiently induced c-fos mRNA. The c-fos mRNA in A5GR cells, however, was not inducible with all treatments, suggesting that one potential mechanism by which the GR restores glucocorticoid-induced growth inhibition in these cells may involve the desensitization of additional 12-O-tetradecanoylphorbol-13-acetate-inducible elements of the fos promoter.