Metal Response Elements Research Articles

Balance between metallothionein and metal response element binding transcription factor 1 is mediated by zinc ions (review).

Metal ion homeostasis and heavy metal detoxification systems are regulated by certain genes associated with metal ion transport. Metallothionein (MT) and metal response element binding transcription factor 1 (MTF‑1) are important regulatory proteins involved in the mediation of intracellular metal ion balance. Differences in the zinc‑binding affinities of the zinc fingers of MTF‑1 and the α‑ and β‑domains of MT facilitate their regulation of Zn2+ concentration. Alterations in the intracellular concentration of Zn2+ influence the MTF‑1 zinc finger number, and MTF‑1 containing certain zinc finger numbers regulates the expression of corresponding target genes. The present review evaluates the association between zinc finger number in MTF‑1 protein, MTF‑1 target genes and the mechanism underlying MT regulation of the zinc finger number in MTF‑1.

Crude oil exposure results in oxidative stress-mediated dysfunctional development and reproduction in the copepod Tigriopus japonicus and modulates expression of cytochrome P450 (CYP) genes

In this study, we investigated the effects of the water-accommodated fraction (WAF) of crude oil on the development and reproduction of the intertidal copepod Tigriopus japonicus through life-cycle experiments. Furthermore, we investigated the mechanisms underlying the toxic effects of WAF on this benthic organism by studying expression patterns of cytochrome P450 (CYP) genes. Development of T. japonicus was delayed and molting was interrupted in response to WAF exposure. Hatching rate was also significantly reduced in response to WAF exposure. Activities of antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were increased by WAF exposure in a concentration-dependent manner. These results indicated that WAF exposure resulted in oxidative stress, which in turn was associated with dysfunctional development and reproduction. To evaluate the involvement of cytochrome P450 (CYP) genes, we cloned the entire repertoire of CYP genes in T. japonicus (n=52) and found that the CYP genes belonged to five different clans (i.e., Clans 2, 3, 4, mitochondrial, and 20). We then examined expression patterns of these 52 CYP genes in response to WAF exposure. Three TJ-CYP genes (CYP3024A2, CYP3024A3, and CYP3027C2) belonging to CYP clan 3 were significantly induced by WAF exposure in a time- and concentration-dependent manner. We identified aryl hydrocarbon responsive elements (AhRE), xenobiotic responsive elements (XREs), and metal response elements (MRE) in the promoter regions of these three CYP genes, suggesting that these genes are involved in detoxification of toxicants. Overall, our results indicate that WAF can trigger oxidative stress and thus induce dysfunctional development and reproduction in the copepod T. japonicus. Furthermore, we identified three TJ-CYP genes that represent potential biomarkers of oil pollution.

Copper transport mediated by nanocarrier systems in a blood-brain barrier in vitro model.

Copper (Cu) is a cofactor of various metalloenzymes and has a role in neurodegenerative diseases with disturbed Cu homeostasis, for example, in Alzheimer's disease (AD) and Menkes disease. To address Cu imbalances, we synthesized two different dendritic nanoparticles (NP) for the transport of Cu(II) ions across the blood-brain barrier (BBB). The synthesized NPs show low toxicity and high water solubility and can stabilize high amounts of Cu(II). The Cu(II)-laden NPs crossed cellular membranes and increased the cellular Cu level. A human brain microvascular endothelial cell (HBMEC) model was established to investigate the permeability of the NPs through the BBB. By comparing the permeability × surface area product (PSe) of reference substances with those of NPs, we observed that NPs crossed the BBB model two times more effectively than (14)C-sucrose and sodium fluorescein (NaFl) and up to 60× better than Evans Blue labeled albumin (EBA). Our results clearly indicate that NPs cross the BBB model effectively. Furthermore, Cu was shielded by the NPs, which decreased the Cu toxicity. The novel design of the core-shell NP enabled the complexation of Cu(II) in the outer shell and therefore facilitated the pH-dependent release of Cu in contrast to core-multishell NPs, where the Cu(II) ions are encapsulated in the core. This allows a release of Cu into the cytoplasm. In addition, by using a cellular detection system based on a metal response element with green fluorescent protein (MRE-GFP), we demonstrated that Cu could also be released intracellularly from NPs and is accessible for biological processes. Our results indicate that NPs are potential candidates to rebalance metal-ion homeostasis in disease conditions affecting brain and neuronal systems.

Activity, regulation, copy number and function in the glyoxalase system

Molecular, catalytic and structural properties of glyoxalase pathway enzymes of many species are now known. Current research has focused on the regulation of activity and expression of Glo1 (glyoxalase I) and Glo2 (glyoxalase II) and their role in health and disease. Human GLO1 has MRE (metal-response element), IRE (insulin-response element), E2F4 (early gene 2 factor isoform 4), AP-2α (activating enhancer-binding protein 2α) and ARE (antioxidant response-element) regulatory elements and is a hotspot for copy number variation. The human Glo2 gene, HAGH (hydroxyacylglutathione hydrolase), has a regulatory p53-response element. Glo1 is linked to healthy aging, obesity, diabetes and diabetic complications, chronic renal disease, cardiovascular disease, other disorders and multidrug resistance in cancer chemotherapy. Mathematical modelling of the glyoxalase pathway predicts that pharmacological levels of increased Glo1 activity markedly decrease cellular methylglyoxal and related glycation, and pharmacological Glo1 inhibition markedly increases cellular methylglyoxal and related glycation. Glo1 inducers are in development to sustain healthy aging and for treatment of vascular complications of diabetes and other disorders, and cell-permeant Glo1 inhibitors are in development for treatment of multidrug-resistant tumours, malaria and potentially pathogenic bacteria and fungi.

Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-1

BackgroundPreviously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion.ResultsUsing enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels.ConclusionsThese studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway allowing the survival of C. intestinalis in continued, elevated presence of metals in the environment.

Characterization of the GufA subfamily member SLC39A11/Zip11 as a zinc transporter

Cellular zinc influx and efflux are maintained by two major transporter families, the ZIP (SLC39A) and ZnT (SLC30A or CDF) molecules. The functions of one molecule in this class, ZIP11/SLC39A11, remain unclear. Bioinformatics analysis of the distribution and evolutionary relationships of different ZIP members in eukaryotes and prokaryotes indicated that Zip11, the sole member of gufA subfamily, is an ancient ZIP family member that might have originated in early eukaryotic ancestors. Murine Zip11 mRNA is abundantly expressed in testes and the digestive system including stomach, ileum and cecum. Analysis of cellular zinc content, metallothionein levels, and cell viability under high or low zinc conditions in cells transfected with a murine Zip11 expression plasmid, suggest that Zip11 is a zinc importer. Further, cellular zinc concentrations and metallothionein levels decreased when Zip11 was knocked down. In mice supplemented with zinc, both mRNA and protein levels of Zip11 were slightly up-regulated in several tissues. The metal response element sequences (MREs) upstream of the first exon of Zip11 responded to elevated extracellular zinc concentrations, as assessed by luciferase reporter assays. Mutagenic analysis showed that several of the MREs could regulate Zip11 promoter activity, and metal-responsive transcription factor-1 (MTF-1) was shown to be involved in this process. Collectively, these data suggest that Zip11 has unique protein sequence and structure features, it functions as a cellular zinc transporter, and its expression is at least partially regulated by zinc via hMTF-1 binding to MREs of the Zip11 promoter.

Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.

Identification and functional characterization of MRE-binding transcription factor (MTF) in Crassostrea gigas and its conserved role in metal-induced response

The full-length cDNA that encodes the MRE-binding transcription factor (MTF) was cloned from the Pacific oyster (Crassostrea gigas) using reverse transcription polymerase chain reaction and the rapid amplification of cDNA ends. The cgMTF cDNA sequence is 2892 bp long, with a 2508 bp open reading frame that encodes an 835-amino acid polypeptide. Multiple alignment revealed that cgMTF has four putative zinc finger-like regions in cgMTF with three C2C2-type zinc fingers and one C2H2-type zinc finger. After 12 h of exposure to Cd(2+), the cgMTF mRNA level was increased in a dose-dependent manner, which then subsided with time. cgMTF stimulates the cgMT promoter reporter in the HEK293 cell line in a dose-dependent manner. When either of the metal-responsive elements (MRE1 or MRE2) of the cgMT promoter was mutated, the cgMT promoter reporter activity was significantly reduced. After the two MREs were mutated simultaneously, the promoter activity was completely abolished. In conclusion, we identified an MTF in C. gigas and revealed the presence of an evolutionarily conserved molecular mechanism for coping with environmental metal stress.

Effective induction of pblac1 laccase by copper ion in Polyporus brumalis ibrc05015

Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.

Natural Variation at the FRD3 MATE Transporter Locus Reveals Cross-Talk between Fe Homeostasis and Zn Tolerance in Arabidopsis thaliana

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator.

Copper induction of enhanced green fluorescent protein expression inPleurotus ostreatusdriven by laccasepoxa1bpromoter

In silico analyses of several laccase promoter sequences have shown the presence of many different responsive elements differentially distributed along the promoter sequences. Analysis of Pleurotus ostreatus laccase promoter poxa1b extending around 1400-bp upstream of the start codon showed the presence of several putative response elements, such as 10 metal-responsive elements. Development of a system for in vivo analysis of P. ostreatus laccase promoter poxa1b by enhanced green fluorescent protein expression was carried out, based on a polyethylene glycol-mediated procedure for fungal transformation. Quantitative measurement of fluorescence expressed in P. ostreatus transformants grown in the presence and in the absence of copper sulfate was performed, demonstrating an increase in expression level induced by the metal.

Prenatal zinc deficiency-dependent epigenetic alterations of mouse metallothionein-2 gene

Zinc (Zn) deficiency in utero has been shown to cause a variety of disease states in children in developing countries, which prompted us to formulate the hypothesis that fetal epigenetic alterations are induced by zinc deficiency in utero. Focusing on metallothionein (MT), a protein that contributes to Zn transport and homeostasis, we studied whether and how the prenatal Zn status affects gene expression. Pregnant mice were fed low-Zn (IU-LZ, 5.0 μg Zn/g) or control (IU-CZ, 35 μg Zn/g) diet ad libitum from gestation day 8 until delivery, with a regular diet thereafter. Bisulfite genomic sequencing for DNA methylation and chromatin immunoprecipitation assay for histone modifications were performed on the MT2 promoter region. We found that 5-week-old IU-LZ mice administered cadmium (Cd) (5.0 mg/kg b.w.) have an elevated abundance of MT2 mRNA compared with IU-CZ mice. Alteration of histone modifications in the MT2 promoter region having metal responsive elements (MREs) was observed in 1-day-old and 5-week-old IU-LZ mice compared with IU-CZ mice. In addition, prolongation of MTF1 binding to the MT2 promoter region in 5-week-old IU-LZ mice upon Cd exposure is considered to contribute to the enhanced MT2 induction. In conclusion, we found for the first time that Zn deficiency in utero induces fetal epigenetic alterations and that these changes are being stored as an epigenetic memory until adulthood.

Enhanced Green Fluorescent Protein Expression in Pleurotus ostreatus for In Vivo Analysis of Fungal Laccase Promoters

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

Metal Dealing at the Origin of the Chordata Phylum: The Metallothionein System and Metal Overload Response in Amphioxus

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.

Molecular cloning and expression of novel metallothionein (MT) gene in the polychaete Perinereis nuntia exposed to metals

To report a novel metallothionein (MT) gene and evaluate its potency as a biomarker, we clone this MT gene and measured the expression levels in the metal-exposed polychaete Perinereis nuntia. Accumulated metal contents and metallothionein-like proteins (MTLPs), which have been recognized as potential biomarkers, were compared with the relative mRNA expressions of the MT gene of P. nuntia (Pn-MT). In addition, the metal-binding affinity was estimated by recombinant Pn-MT protein. Pn-MT having high cysteine residues with three metal response elements in the promoter region closely clusters with those of other invertebrates. The accumulation patterns of metals were dependent on the exposure times in lead (Pb), cadmium (Cd), and copper (Cu) exposure. Particularly, both MTLP levels and relative mRNA expressions of MT were increased with accumulated metal contents and exposure time in P. nuntia exposed to Pb and Cd. There was no significant modulation of the Pn-MT gene in polychaetes exposed to Zn and As. However, the metal-binding ability of the recombinant Pn-MT protein provides a clear evidence for a high affinity of MT to several metal elements. These results suggest that Pn-MT would play an important role in the detoxification and/or sequestration of specific metals (e.g., Pb and Cd) in P. nuntia and have potential as a molecular biomarker in the monitoring of the marine environment using a polychaete.

Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris

The metal response element binding transcription factor-1 (MTF-1) is an important stress response, heavy metal detoxification, and zinc homeostasis factor in eukaryotic organisms from Drosophila to humans. MTF-1 transcriptional regulation is primarily mediated by elevated levels of labile zinc, which direct MTF-1 to bind the metal response element (MRE). This process involves direct zinc binding to the MTF-1 zinc fingers, and zinc dependent interaction of the MTF-1 acidic region with the p300 coactivator protein. Here, the first recombinant expression system for mutant and wild type (WT) mouse MTF-1 (mMTF-1) suitable for biochemical and biophysical studies in vitro is reported. Using the methyltropic yeast Pichia pastoris, nearly half-milligram recombinant WT and mutant mMTF-1 were produced per liter of P. pastoris cell culture, and purified by a FLAG-tag epitope. Using a first pass ammonium sulfate purification, followed by anti-FLAG affinity resin, mMTF-1 was purified to >95% purity. This recombinant mMTF-1 was then assayed for direct protein–protein interactions with p300 by co-immunoprecipitation. Surface plasmon resonance studies on mMTF-1 provided the first quantitative DNA binding affinity measurements to the MRE promotor element (Kd=5±3nM). Both assays demonstrated the functional activity of the recombinant mMTF-1, while elucidating the molecular basis for mMTF-1-p300 functional synergy, and provided new insights into the mMTF-1 domain specific roles in DNA binding. Overall, this production system provides accessibility for the first time to a multitude of in vitro studies using recombinant mutant and WT mMTF-1, which greatly facilitates new approaches to understanding the complex and varied functions of this protein.

Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamyl-cysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.

Epigenetic targets of some toxicologically relevant metals: a review of the literature

The term epigenetics was coined in the context of developmental studies, but the meaning of the term has evolved over time. Epigenetic modulators of gene expression are now known to include DNA methylation, chromatin modifications and noncoding RNAs. The observation that epigenetic changes can be transmitted transgenerationally makes the science of epigenetics very relevant to the field of environmental and molecular toxicology. Heavy metals constitute an important class of environmental contaminants that have been known to influence gene expression directly by binding various metal response elements in the target gene promoters. Recent research suggests that metals can also influence gene expression through epigenetic mechanisms; this adds a new twist to the complexity of metal-mediated gene expression. Here, we review recent studies that investigate the epigenetic, gene expression, and biological effects of various inorganic and organic forms of heavy metals, such as cadmium, arsenic, nickel, chromium, methylmercury, lead, copper and organotin compounds.

Simian Virus 40 Strains with Novel Properties Generated by Replacing the Viral Enhancer with Synthetic Oligonucleotides

Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an "enhancer trap" since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.