Metal Response Elements Research Articles

Plant Metallothioneins: Putative Functions Identified by Promoter Analysis in Silico

Metallothioneins are low-molecular-weight proteins capable of covalently binding heavy metal ions due to the presence of many cysteine residues in their sequences. We analyzed the predicted amino acid sequences of 19 metallothionein (7 from Arabidopsis thaliana and 12 from Oryza sativa) and their promoter sequences in silico in order to determine the potential regulatory cis-elements present in the promoters of metallothionein genes, from which it is possible to determine the putative functions of these genes. The PlantCARE and PLACE databases provided information about the putative regulatory elements in the metallothionein promoters. Metal response element sequences were found in the promoters of eleven O. sativa and two Arabidopsis metallothionein genes. Copper response elements were identified in both model plants, usually in many copies, particularly in O. sativa. Both the high cysteine content and the presence of metal response motifs in the promoters support the suggestion that metallothioneins play a key role in metal detoxification. The most common putative element in the analyzed promoters was CIRCADIAN, which was present in five A. thaliana and eight O. sativa sequences. The methyl jasmonate response sequence, root-specific expression element and drought response element were found only in O. sativa metallothioneins. Light and low temperature response elements, biotic and abiotic stress elements, an abscisic acid-responsive element and an ethylene-responsive element occur in selected metallothionein promoters of both species. A few promoters have putative organ- and cell-specific regulatory elements. The presence of many different motifs in the promoters of the Arabidopsis and O. sativa genes implies that metallothioneins are general stress response proteins with many important functions in plants, including regulation of their normal development and adaptation to changing environmental conditions.

Isoform-specific response of two GAPDH paralogs during bacterial challenge and metal exposure in mud loach (Misgurnus mizolepis: Cypriniformes) kidney and spleen

Gene expression of two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) paralogs was examined during Edwardsiella tarda challenge and heavy metal exposures in mud loach (Misgurnus mizolepis; Cypriniformes) kidney and spleen. Transcription of the two mud loach GAPDH paralogs (mlGAPDH-1 and mlGAPDH-2) was significantly modulated by these stimulatory challenges in an isoform-dependent manner. Based on the real-time RT-PCR analysis, the mlGAPDH-2 transcripts were more preferentially induced by E. tarda challenge, whereas the mlGAPDH-1 transcripts were proven to show more inducibility in response to heavy metal exposure using Cd, Cu, Mn and Zn at <TEX>$5{\mu}M$</TEX>. Their isoform-specific response patterns were closely in accordance with the TF binding profiles in promoter and intron-1 of the two mlGAPDH isoforms, in which the mlGAPDH-2 has more binding sites for immune-related transcription factors than mlGAPDH-1 while the mlGAPDH-1 possesses exclusively metal responsive elements in its intron. Collectively, the mlGAPDHs are potentially involved in cellular pathways independent of glycolysis and the two GAPDH paralogs might undergo functional diversification or subfunctionalization at least at the transcription level.

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copper II

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson's disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu(II)(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu(II)(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu(II)(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu(II)(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from Cu(II)(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O(2) as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu(II)(atsm) that increases cellular retention of the Cu.

Cannabidiol affects the expression of genes involved in zinc homeostasis in BV-2 microglial cells

Cannabidiol (CBD) has been shown to exhibit anti-inflammatory, antioxidant and neuroprotective properties. Unlike Δ9-tetrahydrocannabinol (THC), CBD is devoid of psychotropic effects and has very low affinity for both cannabinoid receptors, CB1 and CB2. We have previously reported that CBD and THC have different effects on anti-inflammatory pathways in lipopolysaccharide-stimulated BV-2 microglial cells, in a CB1/CB2 independent manner. Moreover, CBD treatment of BV-2 cells, was found to induce a robust change in the expression of genes related to oxidative stress, glutathione deprivation and inflammation. Many of these genes were shown to be controlled by Nrf2 and ATF4 transcription factors.Using the Illumina MouseRef-8 BeadChip platform, DAVID Bioinformatics and Ingenuity Pathway Analysis, we identified functional sets of genes and networks affected by CBD. A subset of genes was found to be regulated by the metal responsive element (MRE)-binding transcription factor-1 (MTF-1) and is shown to be related to zinc homeostasis. We found that CBD upregulates the expression of the mRNAs for metallothionein 2 (Mt2), N-myc-downstream regulated gene 1 and matrix metalloproteinase 23 as well as of the zinc transporters ZnT1/Slc30a1 and Zip4/Slc39a4 but downregulates the expression of the mRNA for the zinc transporter Zip10/Slc39a10 as well as for the zinc finger protein 472. Among these genes, ZnT1, Mt2 and the zinc transporters ZIPs are known to function together to control the intracellular zinc concentration.These results show that CBD, but much less so THC, affects the expression of genes involved in zinc homeostasis and suggest that the regulation of zinc levels could have an important role through which CBD may exert its antioxidant and anti-inflammatory effects.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Abstract 353Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD.Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein.In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures:No relevant conflicts of interest to declare.

A conserved cysteine cluster, essential for transcriptional activity, mediates homodimerization of human metal-responsive transcription factor-1 (MTF-1)

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I) and is also involved in the response to hypoxia and oxidative stress. MTF-1 recognizes a specific DNA sequence motif termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. The functional domains of MTF-1 include, besides the DNA-binding and activation domains and signals for subcellular localization (NLS and NES), a cysteine cluster 632CQCQCAC638 located near the C-terminus. Here we show that this cysteine cluster mediates homodimerization of human MTF-1, and that dimer formation in vivo is important for basal and especially metal-induced transcriptional activity. Neither nuclear translocation nor DNA binding is impaired in a mutant protein in which these cysteines are replaced by alanines. Although zinc supplementation induces MTF-1 dependent transcription it does not per se enhance dimerization, implying that actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates MTF-1 only weakly in the cell lines tested, stabilizes the dimer by inducing intermolecular disulfide bond formation and synergizes with zinc to boost MTF-1 dependent transcription.

Determination of bioavailable-zinc from biofortified wheat using a coupled in vitro digestion/Caco-2 reporter-gene based assay

The zinc bioavailability from wheat-Aegilops derivatives and Triticum monococcum low phytic acid (lpa) mutants with varying zinc concentrations was assessed using a novel metal response element (MRE)-binding transcription factor-1 (MTF-1)/MRE luciferase reporter gene based transactivation assay in the coupled in vitro digestion/Caco-2 model. The zinc content in the genotypes studied ranged from 19 to 65μg/g. The bioavailability enhancement of zinc as determined from the luciferase response of MTF1/MRE transfected cells to cooked digests of biofortified derivatives and the lpa mutants was recorded as a 1.2–2.0 fold-increase, corresponding to 1.8–3.3 fold increases in the zinc content over the respective WL711-cultivar and T. monococcum wild-type controls. The strong positive and negative correlations observed for zinc bioavailability with respect to zinc content and phytate:zinc molar ratios in the derivatives (r=0.74 and −0.76) as well as lpa mutants (r=0.89 and −0.94) illustrate the significance of using these parameters in zinc biofortification programmes. Genotypes with phytate:zinc molar ratios of less than 15, with remarkably enhanced zinc bioavailability, were identified. This method could thus serve as a sensitive transactivation assay for a screening approach that could accurately rank bioavailability of zinc from biofortified staples for subsequent animal studies and advance select lines for human studies.

Characterization of MtnE, the fifth metallothionein member in Drosophila

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missing.

MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restriction.

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.

Genome Organization and Transcription Response to Harvest of Two

Metallothioneins are a class of small cysteine-rich proteins that have been associated with increased tolerance to metal and oxidative stresses in animals, plants, and fungi. We investigated a metallothionein-like (mt-like) gene shown previously to be upregulated in fruiting bodies of the fungus Agaricus bisporus in response to post-harvest storage. Analysis of an A. bisporus genomic DNA cosmid library identified two similar mt-like genes (met1 and met2) arranged as a bidirectional gene pair transcribed from the same promoter region. The promoter contained regulatory elements including 9 metal responsive elements and a CAAT box region 220 bp downstream of met1 that showed striking similarity to a feature in Coprinopsis cinerea mt-like gene promoters. Transcriptional analysis showed that both met genes are significantly and rapidly (within 3 hours) upregulated during post-harvest storage and expression is significantly greater in stipe and cap tissues compared with the gills. However, a strong directionality of the promoter was demonstrated, as transcript levels of met1 were at least two orders of magnitude greater than those of met2 in all samples tested.

Metal sensing by MTF1 through its carboxyl‐terminal cysteine residues

Exposure to metals elicits a wide range of adverse effects including cancer, renal failure, neurological disorders, pulmonary fibrosis, and liver damage in animals and humans. Mammalian cells respond to metal challenges by eliminating the metals, antagonizing their effects, and repairing damaged tissues. The metal-activated transcription factor 1 (MTF1) mediates the induction of metallothioneins (MT1 and 2) and other protective proteins/enzymes in response to toxic metals. The mechanism of MTF1 activation by metals has not been well understood. We analyzed the interaction between arsenic (As) and MTF1 for Mt1 induction. Arsenic potently induces Mt1 mRNA expression in many mammalian cells. Induction is dependent upon functional MTF1 as induction is lost in Mtf1 knockout (KO) cells but is restored upon reconstitution with Mtf1. Induction requires the binding of MTF1 to the metal response elements of endogenous Mt1. Induction is markedly enhanced by cycloheximide. Phenylarsine oxide (PAO), which covalently binds to vicinal protein cysteine thiol groups, induces Mt1 with a magnitude of higher potency than that of arsenic. PAO affinity beads effectively pulls down the carboxyl half of MTF1 (MTF1321–675) by binding to a cluster of five cysteine residues near the terminus. Preincubation with As, Cd, Co, Ni, Ag, Hg, and Bi blocks pull-down of MTF1321–675 by PAO beads in vitro and in vivo, indicating binding of the metal inducers to the same C-terminal cysteine cluster as PAO. Finally, deletion of the cysteine cluster or mutation of the cysteine residues abolishes or markedly reduces the transcription activation activity of MTF1 and the ability of MTF1 to restore Mt1 induction in Mtf1 KO cells. Our findings demonstrate that the C-terminal cysteine cluster of MTF1 plays a critical role in metal sensing, activation of MTF1, and transcription of Mt1 through metal-cysteine thiol interaction.

Transcriptional Regulation of the Beta-Synuclein 5′-Promoter Metal Response Element by Metal Transcription Factor-1

The progression of many human neurodegenerative disorders is associated with an accumulation of alpha-synuclein. Alpha-synuclein belongs to the homologous synuclein family, which includes beta-synuclein. It has been proposed that beta-synuclein may be a natural regulator of alpha-synuclein. Therefore controlling beta-synuclein expression may control the accumulation of alpha-synuclein and ultimately prevent disease progression. The regulation of synucleins is poorly understood. We investigated the transcriptional regulation of beta-synuclein, with the aim of identifying molecules that differentially control beta-synuclein expression levels. To investigate transcriptional regulation of beta-synuclein, we used reporter gene assays and bioinformatics. We identified a region −1.1/−0.6 kb upstream of the beta-synuclein translational start site to be a key regulatory region of beta-synuclein 5′-promoter activity in human dopaminergic cells (SH-SY5Y). Within this key promoter region we identified a metal response element pertaining to a putative Metal Transcription Factor-1 (MTF-1) binding site. We demonstrated that MTF-1 binds to this 5′-promoter region using EMSA analysis. Moreover, we showed that MTF-1 differentially regulates beta-synuclein promoter binding site, as well as beta-synuclein mRNA and protein expression. This effect of MTF-1 on expression was found to be specific to beta-synuclein when compared to alpha-synuclein. Understanding the regulation of synucleins and how they interact may point to molecular targets that could be manipulated for therapeutic benefit. In this study we showed that MTF-1 differentially controls the expression of beta-synuclein when compared to its homolog alpha-synuclein. This could potentially provide a novel targets or pathways for therapeutic intervention and/or treatment of synucleinopathies.

ZnT2-overexpression represses the cytotoxic effects of zinc hyper-accumulation in malignant metallothionein-null T47D breast tumor cells

Human breast tumors accumulate abnormally high levels of zinc (Zn). As a result, numerous studies have implicated Zn hyper-accumulation in the etiology of breast cancer. Zinc accumulation can be cytotoxic, therefore cells have Zn-buffering mechanisms, such as metallothioneins (MT) and vesicular sequestration, which tightly regulate Zn homeostasis. The Zn transporter ZnT2 sequesters Zn into intracellular vesicles and thus can protect cells from Zn cytotoxicity. Herein, we report that malignant breast tumor (T47D) cells do not express MT but have ∼4-fold greater Zn levels compared with non-malignant breast (MCF-10A) cells. Zinc accumulation coincided with ZnT2 over-expression and increased vesicular Zn pools. In this study, we hypothesized that ZnT2 suppression would eliminate protection from Zn accumulation and result in cytotoxicity in malignant breast tumor cells. Suppression of ZnT2 significantly increased cytoplasmic Zn pools (1.6-fold) as assessed with a Zn-responsive reporter assay containing four metal response elements (4X-MRE) fused to luciferase. Increased cytoplasmic Zn pools activated apoptosis in a caspase-independent manner. We observed significant generation of reactive oxygen species (ROS) (2.3-fold), lysosomal swelling and cathepsin D leakage in ZnT2-attenuated compared with ZnT2-expressing cells. Most importantly, tumor cell viability and tumor formation were significantly decreased (∼25%) in ZnT2-attenuated cells compared with ZnT2-expressing cells. Our data indicate that ZnT2 over-expression protects malignant MT-null breast tumor cells from Zn hyper-accumulation by sequestering Zn into intracellular vesicles. Moreover, our results implicate Zn compartmentalizing mechanisms as novel targets for breast cancer therapy.

Cloning and sequencing of phenol oxidase 1 (pox1) gene from Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metal-responsive elements (MREs) were determined in the promoter region, where MRE 1, 2 and 3 were located in positions -20, -62 and -389, respectively. Functional TATA consensus sequences were recognized in positions -78 and -245, while CAAT consensus sequence was recognized in position -171. The putative GC boxes consensus sequences were recognized in positions -175 and -344, and xenobiotic-responsive elements (XREs) in positions -100 and -270. The pox1-DNA gene consists of 2656 bp, with the coding sequence being interrupted by 19 introns. The nucleotide sequence of cDNA (pox1-cDNA) was found to contain an ORF of 1590 bp capable of coding for a protein of 529 amino acid residues. The signal peptide was predicted to be 23 amino acids in length using SIGNALP 3.0 program. Northern blot analysis revealed that strong transcriptional induction was observed in the copper-supplemented cultures for pox1 gene. Key words: Pleurotus, cDNA, pox1, gene promoter, putative sequences, northern blot analysis, copper.

Molecular characterization and functions of zebrafish ABCC2 in cellular efflux of heavy metals

Multidrug-resistance associated protein 2 (MRP2/ABCC2) plays crucial roles in bile formation and detoxification by transporting a wide variety of endogenous compounds and xenobiotics, but its functions in zebrafish (Danio rerio) remain to be characterized. In this study, we obtained the full-length cDNA of zebrafish abcc2, analyzed its expression in developing embryos and adult tissues, investigated its transcriptional response to heavy metals, and evaluated its roles in efflux of heavy metals including cadmium, mercury and lead. Zebrafish abcc2 gene is located on chromosome 13 and composed of 32 exons. The deduced polypeptide of zebrafish ABCC2 consists of 1567 amino acids and possesses most of functional domains and critical residues defined in human ABCC2. Zebrafish abcc2 gene is not maternally expressed and its earliest expression was detected in embryos at 72hpf. In larval zebrafish, abcc2 gene was found to be exclusively expressed in liver, intestine and pronephric tubules. In adult zebrafish, the highest expression of abcc2 gene was found in intestine followed by those in liver and kidney, while relative low expression was detected in brain and muscle. Expression of abcc2 in excretory organs including kidney, liver and intestine of zebrafish larvae was induced by exposure to 0.5μM mercury or 5μM lead. Moreover, exposure to 0.125–1μM of mercury or lead also significantly induced abcc2 expression in these excretory organs of adult zebrafish. Furthermore, overexpression of zebrafish ABCC2 in ZF4 cells and zebrafish embryos decreased the cellular accumulation of heavy metals including cadmium, mercury and lead as determined by MRE (metal responsive element)- or EPRE (electrophile response element)-driven luciferase reporters and atomic absorption spectrometry. These results suggest that zebrafish ABCC2/MRP2 is capable of effluxing heavy metals from cells and may play important roles in the detoxification of toxic metals.

LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells

A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.

Cloning and sequencing of phenol oxidase 1 (pox1) gene from Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metalresponsive elements (MREs) were determined in the promoter region, where MRE 1, 2 and 3 were located in positions -20, -62 and -389, respectively. Functional TATA consensus sequences were recognized in positions -78 and -245, while CAAT consensus sequence was recognized in position -171. The putative GC boxes consensus sequences were recognized in positions -175 and -344, and xenobiotic-responsive elements (XREs) in positions -100 and -270. The pox1-DNA gene consists of 2656 bp, with the coding sequence being interrupted by 19 introns. The nucleotide sequence of cDNA (pox1- cDNA) was found to contain an ORF of 1590 bp capable of coding for a protein of 529 amino acid residues. The signal peptide was predicted to be 23 amino acids in length using SIGNALP 3.0 program. Northern blot analysis revealed that strong transcriptional induction was observed in the coppersupplemented cultures for pox1 gene. Key words : Pleurotus, cDNA, pox1, gene promoter, putative sequences, northern blot analysis, copper.

Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells

BackgroundStudies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine.ResultsThe histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1) binding to metal response elements (MRE) elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells.ConclusionThe MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the urothelial cells with either Cd+2 or As+3 modified the chromatin of the MT-3 promoter to a bivalent state of promoter readiness. Urinary cytology for MT-3 positive cells would not improve the diagnosis of urothelial cancer, but might have potential as a biomarker for tumor progression.

Polycomb repressive complex 2 in embryonic stem cells: an overview

Polycomb Group Proteins (PcG) are a family of epigenetic regulators responsible for the repression of an array of genes important in development and cell fate specification. PcG proteins complex to form two types of epigenetic regulators: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). Although the mechanisms regulating PRC2 recruitment and activity in mammals remain poorly understood, recent work has identified a non-canonical PRC2 in mouse embryonic stem cells (mESC) with unique activities required for repression of PRC2 target genes and necessary for mESC differentiation and somatic cell reprogramming. Here we review the functions of PRC2 in embryonic stem cells and explore the role of the newly identified mESC specific PRC2 regulatory subunits Jarid2 (jumonji, AT rich interactive domain 2), Mtf2 (metal response element binding transcription factor 2) and esPRC2p48.

Novel drug targets based on metallobiology of Alzheimer's disease

Importance of the field: Increased localization of Zn, Fe, Cu and Al within the senile plaques (SP) exacerbates amyloid beta (Aβ)-mediated oxidative damage, and acts as catalyst for Aβ aggregation in Alzheimer's disease (AD). Thus, disruption of aberrant metal–peptide interactions via chelation therapy holds considerable promise as a rational therapeutic strategy against Alzheimer's amyloid pathogenesis.Areas covered in this review: The complexities of metal-induced genesis of SP are reviewed. The recent advances in the molecular mechanism of action of metal chelating agents are discussed with critical assessment of their potential to become drugs.What the reader will gain: Taking into consideration the interaction of metals with the metal-responsive elements on the Alzheimer's amyloid precursor protein (APP), readers will gain understanding of several points to bear in mind when developing a screening campaign for AD-therapeutics.Take home message: A functional iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of the APP transcript regulates neural APP translation. Desferrioxamine, clioquinol, tetrathiolmolybdate, dimercaptopropanol, VK-28, and natural antioxidants, such as curcumin and ginko biloba need critical evaluation as AD therapeutics. There is a necessity for novel screens (related to metallobiology) to identify therapeutics effective in AD.