InteractiveFly: GeneBrief

Metal response element-binding Transcription Factor-1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Metal response element-binding Transcription Factor-1

Synonyms -

Cytological map position- 67B5--6

Function - transcription factor

Keywords - heavy metal regulation

Symbol - MTF-1

FlyBase ID: FBgn0040305

Genetic map position - 3L

Classification - zinc fingers, C2H2 type

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Le Manh, H., Guio, L., Merenciano, M., Rovira, Q., Barrón, M.G. and González, J. (2017). Natural and laboratory mutations in kuzbanian are associated with zinc stress phenotypes in Drosophila melanogaster. Sci Rep 7: 42663. PubMed ID: 28218276
Summary:
Stress response to heavy metals is mediated by the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to metal response elements (MREs) and changes the expression of target genes. kuzbanian (kuz), a metalloendopeptidase that activates the evolutionary conserved Notch signaling pathway, has been identified as an MTF-1 target gene. FBti0019170, inserted in a kuz intron, is putatively adaptive transposable element in the Drosophila genome. This study investigated whether a laboratory mutant stock overexpressing kuz is associated with zinc stress phenotypes. It was found that both embryos and adult flies overexpressing kuz are more tolerant to zinc compared with wild-type flies. On the other hand, the effect of FBti0019170 on zinc stress tolerance depends on developmental stage and genetic background. In the majority of the genetic backgrounds analyzed, FBti0019170 has a deleterious effect in unpolluted environments in pre-adult stages.
Qiang, W., Huang, Y., Wan, Z. and Zhou, B. (2017). Metal-metal interaction mediates the iron induction of Drosophila MtnB. Biochem Biophys Res Commun 487(3): 646-652. PubMed ID: 28435068
Summary:
Metallothionein (MT) protein families are a class of small and universal proteins rich in cysteine residues. They are synthesized in response to heavy metal stresses to sequester the toxic ions by metal-thiolate bridges. Five MT family members, namely MtnA, MtnB, MtnC, MtnD and MtnE, have been discovered and identified in Drosophila. These five isoforms of MTs are regulated by metal responsive transcription factor dMTF-1 and play differentiated but overlapping roles in detoxification of metal ions. Previous researches have shown that Drosophila MtnB responds to copper (Cu), cadmium (Cd) and zinc (Zn). Interestingly this study found that Drosophila MtnB expression also responds to elevated iron levels in the diet. Further investigations revealed that MtnB plays limited roles in iron detoxification, and the direct binding of MtnB to ferrous iron in vitro is also weak. The induction of MtnB by iron turns out to be mediated by iron interference of other metals, because EDTA at even a partial concentration of that of iron can suppress this induction. Indeed, in the presence of iron, zinc homeostasis is altered, as reflected by expression changes of zinc transporters dZIP1 and dZnT1. Thus, iron-mediated MtnB induction appears resulting from interrupted homeostasis of other metals such as zinc, which in turns induced MtnB expression. Metal-metal interaction may more widely exist than has been expected.
BIOLOGICAL OVERVIEW

From insects to mammals, metallothionein genes are induced in response to heavy metal load by the transcription factor MTF-1, which binds to short DNA sequence motifs, termed metal response elements (MREs). A novel and seemingly paradoxical role is described for MTF-1 in Drosophila in that it also mediates transcriptional activation of Ctr1B, a copper importer, upon copper depletion. Activation depends on the same type of MRE motifs in the upstream region of the Ctr1B gene as are normally required for metal induction. Thus, a single transcription factor, MTF-1, plays a direct role in both copper detoxification and acquisition by inducing the expression of metallothioneins and of a copper importer, respectively (Selvaraj, 2005).

Copper is an essential trace element that serves as a catalytic cofactor for several enzymes that are mainly involved in respiration, iron transport, and oxidative stress protection (Puig, 2002). However, an excess of copper ions can catalyze cytotoxic reactions; thus, every organism must be able to tightly regulate copper levels. Copper imbalance in humans is the cause of serious diseases, such as Menkes syndrome and Wilson disease, and has also been implicated in Alzheimer's disease and prion-type diseases. Copper homeostasis can be regulated at the level of copper uptake, distribution, chelation, and export. The cellular proteins that are involved in copper homeostasis, such as importers, exporters, and scavengers, are regulated by different mechanisms including transcriptional activation or repression, changes in protein stability, and the modulation of protein trafficking (Selvaraj, 2005).

From insects to mammals, heavy metal detoxification is controlled to a large extent by the zinc finger transcription factor MTF-1 (metal response element-binding transcription factor-1, also referred to as metal-responsive transcription factor, or just metal transcription factor) (Westin, 1988; Radtke, 1993; Langmade, 2000; Giedroc, 2001b; Lichtlen, 2001; Zhang, 2001). Metal response elements (MREs) of consensus TGCRCNC (where R stands for A or G and N for any of the four bases) are cis-regulatory DNA sequences that specifically bind MTF-1 and are essential and sufficient for transcriptional induction upon heavy metal load (Stuart, 1985; Westin, 1988). Major target genes of MTF-1 are the genes encoding metallothioneins—short, cysteine-rich proteins that have the ability to bind and thereby sequester heavy metals (Kägi, 1991; Palmiter 1998). In the mouse, MTF-1 is an essential gene, the knockout of which results in embryonic lethality due to liver degeneration (Günes, 1998). The strong up-regulation of the transcription of metallothionein genes upon heavy metal load is abrogated in MTF-1 knockout cells (Heuchel, 1994; Günes, 1998). A conditional knock-out of MTF-1 in the mouse liver produced no phenotype in normal laboratory conditions, but mice were more susceptible to cadmium toxicity (Wang, 2004). As in the case of mammals, in Drosophila a major function of the MTF-1 (dMTF-1) is in the activation of metallothionein genes in response to heavy metal load (Zhang, 2001). There are four metallothionein genes in Drosophila, each harboring multiple MREs in their enhancer/promoter region. However, unlike the situation in the mouse, knockout of dMTF-1 is not lethal in Drosophila. The mutant flies (dMTF-1140-1R) survive well under laboratory conditions but are extremely sensitive to elevated levels of heavy metals including zinc, copper, and cadmium. Consistent with the phenotype, exposure of dMTF-1 mutants to heavy metal load failed to induce metallothionein genes (Egli, 2003; Balamurugan, 2004) (Selvaraj, 2005).

In light of the established role of MTF-1 under conditions of heavy metal load, it came as a surprise that in Drosophila, MTF-1 mutants also died at larval stages when challenged with nutritional copper scarcity (Egli, 2003). This seeming paradox prompted an investigation of the role of MTF-1 during copper starvation. Microarray analysis and identified the copper importer Ctr1B was identified as a potential target gene of dMTF-1. There are three Ctr-type copper transporters in Drosophila, namely, Ctr1A, Ctr1B, and Ctr1C (Zhou, 2003). Ctr1B function is important during larval stages, where efficient copper uptake is essential for rapid growth. Ctr1B knockout flies (Ctr1B3-4) survive well in normal laboratory conditions but are extremely sensitive to nutritional copper scarcity and, to a lesser degree, also to copper load. The sensitivity of the mutants to copper depletion is consistent with the copper uptake function of Ctr1B. It was speculated (Zhou, 2003) that the sensitivity of the mutants to copper load was due to an inability to mobilize copper to a potential copper-dependent protein or a storage tissue (Selvaraj, 2005).

This study demonstrates that the lethal phenotype of dMTF-1 mutants under copper insufficiency conditions is due to the failure of regulating the copper importer Ctr1B. Interestingly, the upstream regulatory region of the Ctr1B gene contains MREs that conform to the consensus found in metallothionein genes. By genetic and biochemical analyses it was shown that these MREs are, however, not used for induction upon copper load, but are essential for the activation of Ctr1B by dMTF-1 under conditions of copper scarcity. Thus, a novel mechanism is revealed whereby a single transcription factor, dMTF-1, plays a central role in both copper detoxification and acquisition, by directly activating transcription of metallothioneins and a copper importer, respectively (Selvaraj, 2005).

It was known from the dMTF-1 knockout study that the Drosophila larvae were not only sensitive to excess copper, zinc, and cadmium but also highly sensitive to copper depletion, as tested by supplementing the food with the specific copper chelator bathocuproinedisulfonate (BCS) (Egli, 2003). To understand this phenotype, the transcriptome response in a deletion mutant of the heavy metal regulator dMTF-1 (dMTF-1140-1R) was assessed. A comparison of microarray data from the dMTF-1 mutant and wild type (WT) larvae revealed that transcripts of one of the copper importers, Ctr1B, were reduced in the dMTF-1 mutant, whereas expression of the related genes Ctr1A and Ctr1C was not affected. A microarray analysis of genes up-regulated in low copper conditions in wild-type Drosophila, in one case, and genes with decreased expression in the dMTF-1 deletion mutant in normal food, in the other Ctr1B as the only overlapping gene. These findings were confirmed by RNA blotting, which showed in wild-type Drosophila, an opposite regulation of the Ctr1B gene as compared with a well-characterized target gene of MTF-1, metallothionein A (MtnA). While the latter was strongly induced by excess copper in the food, Ctr1B was at the same time down-regulated, but induced by copper chelator treatment. In the dMTF-1 mutant, the MtnA transcripts were not detectable at any condition, while Ctr1B transcripts were reduced in normal food and could no longer be up-regulated in response to copper chelator treatment (Selvaraj, 2005).

The loss of regulation of Ctr1B in the dMTF-1 mutant prompted a test to see whether Ctr1B was responsible for the unexpected sensitivity to copper deprivation of the dMTF-1 mutant Drosophila. For this, attempts were made to shortcut the regulation by constitutively overexpressing Ctr1B. Several transgenic fly lines were generated with a Ctr1B ORF driven by UASGAL. The Ctr1B transgenic flies survived well but invariably died if crossed with flies constitutively expressing the Gal4 transcription factor via the actin5c promoter. To test whether this lethality was due to uncontrolled copper import or another effect, the larvae were raised in food with increasing amounts of copper chelator. The results were clear, in that the flies survived only in the presence of high chelator concentrations, while wild-type flies survived under all conditions. These observations suggest that the larvae died from copper toxicosis, even in normal food, due to the strong, ectopic expression of Ctr1B. The same system was used to test whether this constitutive expression of Ctr1B could rescue the lethal phenotype of the dMTF-1 mutant under low copper conditions. dMTF-1 mutant Drosophila are developmentally arrested and die at second or third instar larval stages when the concentration of BCS reaches 50 µM in the food. Strikingly, constitutive Ctr1B expression rescued the developmental arrest and larval lethality of the dMTF-1 mutants under copper depletion, and several viable dMTF-1 mutant flies were obtained from food containing 50 or even 100 µM BCS. The rescued dMTF-1 mutant flies were normal and fertile. The constitutive Ctr1B expression and lack of tissue specificity probably prevented a complete rescue of dMTF-1 mutants in all concentrations of BCS tested (Selvaraj, 2005).

While these results demonstrated that Ctr1B is an essential downstream target gene of dMTF-1 under copper starvation, the question remained whether the response is direct or indirect. Inspection of the upstream sequences of the Ctr1B gene revealed a cluster of three metal response elements, designated MRE1-MRE3, and a fourth one set apart from them. To determine the significance of these MREs, a comparison was made to the several related species of Drosophila whose genome sequences are available in the database. The Ctr1B genomic region from Drosophila virilis was amplified and sequenced and these data were included in the comparison. While the majority of upstream sequences have diverged considerably, the MRE cluster is highly conserved, both regarding the MREs themselves and their flanking sequences, among the four species. The comparisons also revealed that the fourth MRE, which lacks the typical flanking sequences of good MREs, is not conserved in the other Drosophila species. To test whether dMTF-1 can bind to the MREs of Ctr1B, electrophoretic mobility shift assay (EMSA) experiments were conducted. Drosophila S2 cells were transfected with either Drosophila MTF-1 or human MTF-1 expression plasmids, and extracts from these cells were tested with radiolabeled oligonucleotides containing MREs from the Ctr1B upstream region. Indeed, both MRE1 and an oligo containing the closely spaced MRE2 and MRE3 of Ctr1B bind strongly to dMTF-1 and hMTF-1 and are well comparable to the binding of a consensus oligo designated MRE-s (Selvaraj, 2005).

To narrow the region responsible for copper regulation, transgenic flies were tested with deletion constructs driving a fluorescent protein reporter. In one of these, the EGFP coding sequence was fused to the last codon preceding the stop codon of Ctr1B, thereby preserving not only the coding sequence but also the introns that might harbor regulatory sequences (AH3). In another construct, the first codon of Ctr1B was fused to EGFP (AH2). Transgene expression was found to be strongly induced in the larval gut by BCS-supplemented food. Consistent with the role of Ctr1B in copper import, plasma-membrane-localized green fluorescence was observed in the cells of the larval gut of AH3 transgenic flies. Removal of the Ctr1B upstream region harboring the MRE1-MRE3 cluster (AH1) had a dramatic effect, in that the reporter gene was no longer inducible by copper depletion. The quantification of the EGFP transcripts from whole larvae revealed a two- to threefold up-regulation of transcription in BCS-containing food. In line with a role of MTF-1 in Ctr1B regulation, there was no green fluorescence from AH2 and AH3 transgenes in the gut of dMTF-1 knockout larvae. A genomic deletion of the Ctr1B locus was generated by imprecise excision of an adjacent P element. One deletion of 685 bp including the region that harbors the MREs was recovered. This Ctr1B allele, designated Ctr1B11-19, was no longer induced by copper starvation. For further elucidation of the role of MREs, several transgenic fly lines were constructed that contained point mutations in individual Ctr1B promoter MREs. The results with transgenic larvae showed that MREs are, indeed, critical for the up-regulation of Ctr1B transcription under copper limiting conditions; these specific mutations abolished the expression in low copper, indistinguishable from a deletion of the entire cluster. Even the mutation of a single motif (MRE1) had the same detrimental effect (Selvaraj, 2005).

To assess the biological importance of MREs in Ctr1B gene regulation, the ability of the Ctr1B constructs to rescue Ctr1B-null mutant flies in low and high copper concentrations was tested. The results confirm the importance of the MREs in the Ctr1B gene in that only the Ctr1B-EGFP construct with the wild-type promoter (AH3), but none of the constructs with MRE mutations, rescued the Ctr1B-null mutants from lethality in low copper. These results lend further credence to a scenario in which Ctr1B gene transcription is induced upon copper depletion via upstream MRE sequences and transcription factor MTF-1. As mentioned above, Ctr1B-null mutants are also more sensitive to copper load than wild type. The exact reason for this remains to be elucidated; in any case, it was found that the high-copper sensitivity can be rescued to a large extent even by a Ctr1B transgene lacking the triple MREs. Thus, the main role of these MREs is in copper scarcity, rather than copper load (Selvaraj, 2005).

The results obtained so far demonstrate that dMTF-1 is not only essential for the activation of metallothioneins and other target genes upon heavy metal load, but also to regulate transcription of Ctr1B under copper starvation. Because in the case of other transcription factors, subtle differences in the DNA-binding site can determine whether the factor interacts with a coactivator or a corepressor, it is considered possible that the MRE motifs of Ctr1B themselves could bring about transcriptional induction at low copper. To this end, two types of reporter transgenes were generated with a synthetic 'minipromoter', one containing the four MRE motifs from the Ctr1B gene with hardly any intervening sequences, arranged in tandem arrays, and another one where only MRE1 was multimerized to four copies. These two reporter transgenes were compared with a similar synthetic minipromoter, which contains a tandem array of MRE motifs derived from the metallothionein B (MtnB) gene (Zhang, 2001). Interestingly, all three reporter transgenes behaved like a genuine metallothionein promoter: They were strongly induced when the larvae were fed with copper, but were not responsive to low copper. Also in cell culture, all three reporter constructs were robustly induced by copper treatment. Thus, the Ctr1B MREs on their own are not sufficient to confer transcriptional induction upon copper depletion, but rather respond to metal load. This suggests that sequences in addition to MREs in the Ctr1B enhancer/promoter region contribute to the regulatory characteristics of that gene (Selvaraj, 2005).

How could one transcription factor exert two diametrically different functions? One possibility could be that this special architecture of the MREs in the Ctr1B enhancer/promoter facilitates cooperative binding between dMTF-1 and a hypothetical copper-dependent repressor protein. Under normal conditions, this repressor would be partially removed, resulting in a moderate expression, while under copper starvation it would dissociate from dMTF-1 completely, yielding higher expression. Further experiments will be required to elucidate the exact mechanism of Ctr1B activation via dMTF-1 under conditions of copper starvation (Selvaraj, 2005).

How do other organisms handle copper excess and copper starvation? In the yeast Saccharomyces cerevisiae, the two extremes require different transcription factors. The homologs of Ctr1 that import copper are activated upon copper starvation by the Mac1 transcription factor; the activation of metallothionein genes upon copper load is driven by the transcription factor Ace1. In mammals, there are two Ctr homologs, Ctr1 and Ctr2. Neither of them is apparently regulated at the level of transcription by copper availability, and there are no MREs in their enhancer/promoter region (Selvaraj, 2005 and references therein).

In conclusion, the major role of MTF-1 is to handle heavy metal load; accordingly, MREs are found in the metallothionein genes and other metal-responsive genes from insects to mammals. In contrast, regulation of the Ctr1B copper importer via MREs/MTF-1 appears to have evolved specifically in Drosophilidae as an efficient way to cope with copper starvation. This represents a novel regulatory mechanism in which one and the same transcription factor serves as an activator of different genes in response to opposite environmental conditions (Selvaraj, 2005).


REGULATION

Transcriptional Regulation

Free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and metallothioneins regulate their own expression by a negative feedback loop

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. This study reports the generation of a metallothionein gene family knockout in Drosophila by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, different metallothioneins were found to be expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's 'copper cells.' The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, it is concluded that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. It is concluded that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop (Egli, 2006a).

The genomic arrangement of the metallothionein genes made the generation of a quadruple knockout a difficult endeavor. All four genes are located on the same chromosome, but they are interspersed with many other genes, which precluded the removal of a large segment. Rather, the genes had to be mutated one by one by means of targeted gene disruption. Drosophila metallothioneins have an important role in copper homeostasis, as well as in the detoxification of cadmium. While cadmium toxicity is often caused by industrial pollution, copper is an essential trace element, and its homeostasis is physiologically important. The copper tolerance of wild-type flies is truly remarkable. In metallothionein mutants, a slight increase in copper levels, whether from a constant exposure or a transient copper shock, results either in larval death or in a shortened life span. With increased sensitivity to copper and cadmium and, to a lesser extent, to zinc, metallothionein mutants mirror most but not all aspects of the Drosophila MTF-1 mutant phenotype. Flies lacking MTF-1 are more sensitive to zinc, silver, and mercury than metallothionein mutants. This difference can best be explained by MTF-1-dependent activation of genes involved in metal homeostasis other than the metallothioneins. For example, MTF-1 induces the expression of a putative zinc exporter (CG3994) under conditions of zinc excess, preventing zinc overload of the cell (Yepiskoposyan, unpublished, reported in Egli, 2006). The sensitivity of MTF-1 mutants to silver might be due to low expression levels of the copper importer Ctr1B, another MTF-1 target gene. Silver exerts its toxicity by competing with copper, and competition is expected to be more severe in MTF-1 mutants, which have lower copper levels than wild-type flies (H. Yepiskoposyan and K. Balamarugan, unpublished data, reported in Egli, 2006a). Besides copper and cadmium, mercury, silver, and zinc induce metallothionein transcription, but these metals are not more toxic to metallothionein mutants than to the wild type. Apparently, metallothioneins are not able to protect against all compounds that induce their synthesis (Egli, 2006a).

Wild-type flies can develop at a copper concentration at least 200-fold higher (1 mM) than the normal copper content in food (5 µM) without survival to adulthood being affected. Indeed for most organisms, copper is a relatively benign trace element, thanks to sophisticated transport and detoxification systems. However, it can be toxic under special circumstances, such as in association with genetic defects of copper homeostasis or upon environmental accumulation, notably in vineyards where it is used as an antifungal agent. Metallothioneins protect the cells from toxic effects of copper by binding and sequestering the metal inside the protein. This metallothionein-copper complex can be conveniently observed in vivo as an orange copper luminescence. The reduction of copper cell luminescence in qMtn mutants suggests that copper is now solvent accessible and able to damage the cell, possibly via the generation of ROS and/or ectopic binding to protein sulfhydryl groups. Formation of ROS by copper is often proposed as the major mechanism of copper toxicity. The sensitivity of SOD1 mutant flies to high levels of copper is in agreement with such a scenario. In the Long-Evans cinnamon rat strain, a model system for a human copper homeostasis disorder, Wilson's disease, copper accumulation in the liver induces ROS production, which results in lipid peroxidation, impaired mitochondrial function, and increased DNA damage. However, under physiological conditions, copper also has a role in antioxidant defense as an essential component of the Cu, Zn-SOD. Low dietary copper levels were found to impair the catalytic function of Drosophila Cu,Zn-SOD, similar to what has been observed with mammals, and such flies display a dramatically shortened life span. The effect of copper concentration on life span apparently follows a U-shape curve, since both copper starvation and elevated concentrations shorten the life span. In both extremes, a likely cause of the premature death of adult flies is the accumulation of ROS-mediated damage. Under conditions of copper load, the shortened life span is particularly evident for metallothionein mutant males that normally express metallothionein mRNA at higher levels than females. Thus, it is proposed that adult males depend more on the protective effects of metallothioneins than females (Egli, 2006a).

All four Drosophila metallothioneins and even a transgene with a synthetic minipromoter composed merely of four tandem copies of MREs are expressed in a very similar tissue- and cell-type-specific pattern in the intestine, which reflects the known accumulation of copper or cadmium. For example, metallothioneins are expressed in the 'copper cells,' which are well known for their peculiar ability to accumulate large amounts of copper. Cadmium has been shown to accumulate in the anterior midgut, in the 'iron cell' region, and in the posterior midgut in a pattern highly similar to the metallothionein induction reported in this study. Since MTF-1 is the only transcription factor known to bind directly to MREs but ubiquitous overexpression of MTF-1 does not change the expression pattern of metallothioneins, it is concluded that metallothionein expression is governed by metal distribution. Thus, a metallothionein promoter driving a fluorescent protein reporter can be used as a highly sensitive and semiquantitative biomarker for cellular metal transport. This may help to investigate the function of putative metal transporters in vivo, as has been demonstrated in a study of the copper exporter ATP7 (Egli, 2006a).

Metallothioneins, together with glutathione, an antioxidant with relatively nonspecific metal binding ability, constitute the first line of defense against the toxic effects of heavy metals in both mammals and insects. As ingested metals first reach the gut cells, metallothioneins in the gut probably serve to trap toxic metals and limit their distribution throughout the body. An interesting example of such trapping of toxic metal is the zinc treatment of Wilson's disease patients, who suffer from copper accumulation in the liver. Zinc treatment induces metallothionein synthesis in the intestine; due to the metallothionein's high affinity to copper, the latter is trapped within intestinal cells and eventually excreted (Egli, 2006a).

A remarkable finding in the present study is the autoregulation of metallothionein expression. Metallothionein promoters used as a reporter of MTF-1 activity are more active in metallothionein mutants. This is not due to a higher copper content, since total body copper does not differ between wild-type and metallothionein mutants. Rather, metallothioneins can inhibit their own expression by inhibiting MTF-1 function via the binding of free metal, which otherwise would directly or indirectly activate MTF-1. The nature of the signal that activates MTF-1 in vivo is still not established. While zinc can directly bind to MTF-1, copper and cadmium interfere with DNA binding of MTF-1 in vitro. They are thus thought to activate MTF-1 indirectly and were indeed shown to do so in a cell-free model system by displacement of zinc from metallothioneins, which in turn binds to MTF-1. The present study, however, shows that metal-loaded metallothioneins are not essential in vivo for the activation of MTF-1 by any of the heavy metals tested. It is therefore likely that zinc can be displaced from other cellular pools. Furthermore, as shown with mammals, MTF-1 regulation in vivo occurs at multiple levels that include nucleocytoplasmic transport, protein phosphorylation, and a conspicuous cysteine cluster toward the C terminus of MTF-1, which might act as a metal sensor. In conclusion, the present study does not support the concept of metallothioneins playing an important role in copper uptake and intracellular distribution but firmly establishes their importance for coping with metal fluctuations, notably for copper homeostasis and cadmium detoxification (Egli, 2006a).

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion were determined. Furthermore, the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant was analyzed. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in this study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification (Yepiskoposyan, 2006).

The imbalance of metals due to genetic disorders or environmental pollution is a worldwide problem associated with adverse health effects. This study unveils a number of genes and regulatory mechanisms involved in maintaining heavy metal homeostasis. The metals tested in this study, cadmium, copper and zinc, induce a wide range of responses, including induction of genes coding for metallothioneins, transporters, components of the glutathione-mediated detoxification pathway, antimicrobial peptides, ubiquitin-conjugating enzymes, heat shock proteins and cytochrome P450 enzymes. The universal induction of metallothioneins by all three metals and the downregulation by copper depletion is in line with their metal scavenger function and protective role against metal toxicity. However, there are differences in the strength of the response of individual metallothioneins: MtnA induction is more prominent in copper, whereas MtnB and MtnC are more strongly induced by zinc and cadmium. Indeed, a recent study suggests that individual metallothioneins preferentially defend against specific metals, with MtnA playing a major role in handling copper excess, MtnB during cadmium and zinc excess, while MtnC and MtnD play only minor roles in defending against these three metals. In absolute terms, expression of MtnA is higher than that of other metallothioneins, with an impressive basal level and consequently a lesser fold-induction, suggesting MtnA to be the major metal scavenger at Drosophila larval stage (Yepiskoposyan, 2006).

Another essential system for metal ion homeostasis is metal export and import. Although transcriptional control is only one of several regulatory processes, numerous changes could be followed in characterized, as well as putative, Drosophila metal transporters in the microarray experiment. To provide adequate intracellular copper concentrations, the transcripts of the high-affinity copper importer Ctr1B are strongly induced by copper depletion; conversely, they are downregulated at high Cu concentration in order to reduce copper uptake. Two zinc transporter genes (ZnT35C and ZnT63C), both encoding ZnT family members involved in zinc efflux, as well as two ATPase transporters (CG6263 and CG18419) presumably involved in cation and lipid transport, were upregulated by Zn and Cd treatments. Of note, several ubiquitin-conjugating enzymes (Ubc) were induced by zinc. In this context it is worth mentioning that the yeast zinc importer Zrt1p is ubiquitinated and subsequently degraded upon zinc load. The upregulation of Ubc might also help to degrade misfolded proteins which are probably formed upon metal load (Yepiskoposyan, 2006).

The results indicate that the larval transcriptome responses to zinc and cadmium share several features. Even though they exert different biological effects, the chemical properties of Zn and Cd are similar, thus part of the regulatory mechanisms to cope with excess zinc are also functional in cadmium stress. Another example of the similarity between the zinc and cadmium responses is the specific and strong induction of two members of the glutathione S-transferase Delta class genes. There are more than 40 GST genes in Drosophila grouped into six classes. Three GSTs, CG17524, CG6776 and CG1681 with homology to epsilon, omega and theta class GSTs, respectively, are preferentially upregulated by zinc. GSTs catalyze the conjugation of glutathione (GSH) to a variety of harmful compounds. The resulting glutathione conjugates (GS-X) can then be sequestered by the vacuole or excreted from the cell by ATP-dependent GS-X pumps. The details of this process vary with different compounds and in different species. In S.cerevisiae, e.g. cadmium ions are conjugated to GSH and the Cd·GS2 conjugate is transported to the vacuole by YCF1, an ATP-binding cassette transporter. It is plausible that the GSTs induced in these studies catalyze the conjugation of Cd and/or Zn to GSH, which eventually will be removed from the cytoplasm. Alternatively, or in addition to conjugating metals, they might conjugate ROS which can occur as byproducts of heavy metal metabolism. Interestingly, a specific ATP-binding cassette transporter CG10505, that shares 26% identity to YCF1, is strongly upregulated by all tested metals. It is tempting to speculate that CG10505 acts in a similar fashion to its yeast counterpart in exporting the metal-GSH conjugates. Nevertheless, the metal specificity of Drosophila CG10505 differs from that of YCF1 because the former contributes to elevated resistance to zinc and copper, rather than to cadmium. Another enzyme involved in GSH conjugate metabolism and excretion, gamma-glutamyl transferase, which is an integral part of the gamma-glutamyl cycle involving the degradation and neo-synthesis of GSH was also induced by zinc in Drosophila. Taken together, these data suggest that GSH conjugation plays a substantial role in the response of Drosophila to metal stress. Support for such a scenario comes from a recent study in the mouse showing the existence of two branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, and the other via glutathione. In agreement with this concept the data indicate that when the MTF-1-dependent response is abrogated, the glutathione-associated pathway is upregulated to compensate for this compromised genetic background (Yepiskoposyan, 2006).

The knockout of the Drosophila MTF-1 gene altered the expression of more than 50 genes in the transcriptome assay. However, there is no obvious phenotype unless the animal is subjected to metal stress: copper, zinc, cadmium and mercury load, or copper starvation. The copper importer Ctr1B and metallothionein genes are well-established targets of Drosophila MTF-1. The Ctr1B mutant is sensitive to changes in copper concentration and the KO of the Drosophila metallothionein family is sensitive to cadmium and copper load; however, these mutants hardly show any phenotype upon zinc challenge. A major role is suggested in zinc detoxification for several other genes (Yepiskoposyan, 2006).

Next to the ABC transporter CG10505, zinc exporter Zn35C contributes to zinc homeostasis in that the truncation mutant ZnT35Cmut is extremely sensitive even to a mild zinc load of 2 mM, while wild-type tolerates up to 14 mM zinc excess. Also, an overexpression of this transporter confers resistance to zinc and can partially rescue the zinc sensitivity of MTF-1 knockout flies. Given the strong zinc sensitivity phenotype of the mutant, it is proposed that ZnT35C is the major transporter of cellular zinc, similar to the mammalian zinc exporter ZnT1. It remains to be seen whether the other zinc exporter-like genes annotated in the Drosophila genome transport zinc from the cytoplasm to vesicular compartments, as do several of the mouse zinc transporters, such as ZnT2, 3, 5 and 7. Drosophila ZnT35C localizes to the plasma membrane both at normal and elevated zinc concentrations; besides, mutant flies have a high zinc content, in support of a role in zinc excretion. It is noted that the mouse zinc exporter ZnT1 was also found to be induced both by cadmium and zinc treatment in an MTF-1-dependent fashion similar to ZnT35C. Thus not only the regulation of metallothioneins but also that of zinc transporters is conserved between insects and mammals. In other cases, metal responses of Drosophila and mammals diverge: the fly orthologs of the mouse cEBP alpha, ndrg1 or klf4 were unchanged in the microarray experiments (Yepiskoposyan, 2006).

An unexpected result of the present study is the induction of ferritin heavy and light chain homologue genes by heavy metals. Ferritin is well known to detoxify, store and transport iron. It was also shown to bind other metal ions and has been speculated to function as a general metal detoxicant in mammals. The results suggest an induction of Drosophila ferritins by metals other than iron, notably zinc. The latter induction depends on MTF-1, based on the following findings: (1) the presence of multiple MREs, (2) the loss of zinc induction in the MTF-1 mutant, and (3) elevated ferritin transcripts upon MTF-1 overexpression. In contrast, iron inducibility must be regulated independently since it is not altered by the absence of MTF-1. In line with different signaling pathways responding to iron load versus excess of zinc, copper or cadmium, it is noted that the classical MTF-1 dependent MtnA gene is not inducible by iron. Yet these pathways apparently overlap in the ferritin genes (Yepiskoposyan, 2006).

The four members of the Drosophila metallothionein family exhibit distinct yet overlapping roles in heavy metal homeostasis and detoxification

Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA, MtnB, MtnC, MtnD, all of which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. This study shows, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the individual metallothionein mutants, the most prominent distinction between them was that MtnA-defective flies were the most sensitive to copper load, while MtnB-defective flies were the most sensitive to cadmium. Using various reporter gene constructs and mRNA quantification, the MtnA promoter is shown to be preferentially induced by copper, while the MtnB promoter is preferentially induced by cadmium. Such a metal preference is also observed at the protein level as the stoichiometric, spectrometric and spectroscopic features of the copper and cadmium complexes with MtnA and MtnB correlate well with a greater stability of copper-MtnA and cadmium-MtnB. Finally, MtnC and MtnD, both of which are very similar to MtnB, display lower copper and cadmium binding capabilities compared to either MtnA or MtnB. In accordance with these binding studies, Drosophila mutants of MtnC or MtnD have a near wild type level of resistance against copper or cadmium load. Furthermore, eye-specific over-expression of MtnA and MtnB, but not of MtnC or MtnD, can rescue a 'rough eye' phenotype caused by copper load in the eye. Taken together, while the exact roles of MtnC and MtnD remain to be determined, the preferential protection against copper and cadmium toxicity by MtnA and MtnB, respectively, are the result of a combination of promoter preference and metal binding (Egli, 2006b).

Coactivator cross-talk specifies transcriptional output

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. RNA interference (RNAi) was used in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. The robust response of the metallothionein genes to heavy metal was used as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, it was found that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a 'checkpoint complex' for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, it was found that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation (Marr, 2006).

There are four known metallothionein genes in Drosophila: MtnA, MtnB, MtnC, and MtnD. Of these, the best characterized is the MtnA gene, which produces a transcript of ~600 bases in length, bearing one intron. All of the regulatory elements required for robust response to heavy metals, including copper, lie within 500 bp of the transcription start site. The gene is controlled by a single activator, metal response element-binding transcription factor 1 (MTF-1), which binds two adjacent metal response elements (MRE) 50 bp upstream of the TATA-box (Zhang, 2001). Quantitative PCR (qPCR) analysis of the endogenous gene in Drosophila S2 cells shows that the gene is highly induced (~250-fold) after a short exposure to copper. The total amount of stable MtnA mRNA approximates the level of the abundant transcript for the ribosomal subunit Rp49. Primer extension analysis confirms that transcriptional activation of the endogenous MtnA gene originates from a unique start site overlapping the core promoter. The transcript accumulates linearly for ~12 h, thus measurements in this time window likely reflect relative levels of transcription of the MtnA gene. Importantly, induction at the endogenous chromosomal locus is easily assayed in order to measure physiologically relevant transcriptional activation in the context of native chromatin. Taken together, these properties establish the endogenous MtnA gene as a useful model for studying transcriptional mechanisms governing an inducible gene (Marr, 2006).

Using chromatin immunoprecipitation (ChIP), it was found that the sequence-specific DNA-binding protein MTF-1 is specifically recruited to the MtnA promoter region in response to copper. Curiously, the ChIP of the promoter region was compared to a region 1 kb downstream, a significant amount of MTF-1 was found to be present on the promoter even in the absence of added copper. Under these conditions, little transcription is detected from this gene. As a preliminary experiment to investigate a potential functional interaction between TFIID and MED, it was first asked whether the two complexes are both recruited in a signal-dependent manner to the MtnA gene. Using ChIP, it was found that both TBP and the TAFs are efficiently recruited to the promoter region in response to copper. In addition, the MED17, MED24, MED26, and MED27 subunits of MED are all recruited to the promoter region in response to copper treatment. Consistent with the high level of induction, RNAPII occupancy at the MtnA promoter is also increased in response to heavy metal treatment. Thus, both core coactivator complexes and RNAPII are efficiently recruited to the promoter region upon induction and resultant binding of MTF-1 to the MREs (Marr, 2006).

Because the ChIP assay is limited to measuring response in a heterogeneous population of cells, a transgenic model system was extablished in Drosophila S2 cells in order to visualize the response at the single-cell level. Such an approach has proved useful in understanding transcription factor dynamics in vivo. By selecting for stably transfected MtnA firefly luciferase reporters, a concatenated transgenic locus was generated in a clonal line of S2 cells. The transgenic locus was assayed for dependence on copper using a luciferase assay. Importantly, transcription initiates a unique site that maps to the correct start site of the MtnA core promoter. With this substantial increase in gene number (~2000) at the integrated transgenic locus, it should now be possible to visualize direct recruitment of specific transcription factors to the MtnA promoter within a single cell (Marr, 2006).

As expected, in the absence of heavy metal, MTF-1 is predominantly cytoplasmic; however, in agreement with ChIP data, some MTF-1 can be detected at the transgenic cluster even in the absence of a metal stimulus. Thus, antibody labeling of MTF-1 provides a useful marker for the subnuclear location of the transgene cluster in both induced and uninduced cells. Notably, the locus is not undergoing transcription (as detected by RNA FISH) in the absence of heavy metal induction despite the presence of some MTF-1 at the transgene cluster. Upon copper induction, MTF-1 vacates the cytoplasm and accumulates selectively at the transgenic locus. Under these same conditions, TBP is also actively recruited to this cluster. Consistent with not only TBP but holo-TFIID complex recruitment, it was found that TAF2 also accumulates at the transgene. Likewise MED components recruited to the transgene were detected using antibodies against MED26. As expected, RNAPII is recruited to the cluster in a copper-dependent manner consistent with the transcriptional induction of the transgene under these conditions. In contrast, TBP-related factor 1 (TRF1), a subunit known to be a key component of the RNA polymerase III core promoter recognition complex, is not recruited to the transgene. This negative control helps rule out the possibility that the tandemly reiterated transgene is simply nonspecifically attracting transcription factors (Marr, 2006).

Having established by two independent methods that both TFIID and MED complexes are recruited to the MtnA promoter in an activator-dependent manner, their role in potentiating transcriptional activation of the endogenous MtnA gene was investigated. The efficient technique of RNAi in Drosophila S2 cells was used to knock down expression of TFIID and MED subunits. In addition, the activator MTF-1 was knocked down to ascertain the extent of the activator’s role in induction. After treatment with copper, total RNA was purified from dsRNA treated and untreated S2 cells and then they were assayed by two independent methods. First, a primer extension analysis was used on equivalent amounts of total RNA. This assay revealed that an accurate transcription is detected from one distinct core promoter start site. Next, qPCR normalized to the Rp49 mRNA was used, to confirm that there is little or no global disturbance of RNAPII transcription (Marr, 2006).

Not surprisingly, depletion of MTF-1 severely reduced transcriptional activation from the MtnA promoter, confirming the central role of this activator. RNAi directed against TBP also had a dramatic inhibitory effect. The MtnA promoter is <10% as active when TBP levels are severely depleted. Surprisingly, knockdown of multiple TAFs had little apparent effect on the ability of MTF-1 to activate MtnA. Indeed, depletion of the TAFs actually stimulated (1.5- to 2-fold) production of RNA. With the exception of TAF11, a reduction of individual TAFs resulted in a remarkably uniform response. The reason for this uniformity became apparent when the stability of the TFIID complex was examined in the RNAi-treated cells. The overall stability of the holo-TFIID complex appears to be coupled to the stability of certain individual TAFs. In the most dramatic example, RNAi-targeted reduction of TAF4 leads to the concomitant loss of TAF1, TAF5, TAF6, and TAF9, as well as a detectable reduction in TBP. Interestingly, TAF2 and TAF11 are largely unaffected by depletion of TAF4. Similar results are observed for the other TAFs as well. When the transcript levels of the TAFs were measure after RNAi treatment, it is clear that the loss of stability occurs at the protein level, since the transcript levels for nontargeted TAFs are unaffected. For example, when TAF4 is targeted, only the TAF4 transcript is depleted (Marr, 2006).

In contrast to the TAFs, RNAi reduction of MED subunits gave striking but variable effects on the ability of MTF-1 to activate transcription from the MtnA promoter. Unlike TFIID, the response is far from uniform. For example, dsRNA directed against MED23 has little effect on induction of MtnA, while loss of MED17, the Drosophila SRB4 homolog, has a strong inhibitory effect. The lack of a uniform response in the MED RNAi led to a further investigation of the potential differential response upon depletion of MED subunits at related promoters activated by MTF-1. As discussed above, Drosophila has four metallothionein genes that respond to heavy metals. Three of these—MtnA, MtnB, and MtnD—are active in S2 cells. All three of these genes are specifically activated by the same factor, MTF-1. All three Mtn genes were examined in a single experiment using qPCR. First, it was confirmed that all three promoters, MtnA, MtnB, and MtnD, require MTF-1 for induction. Remarkably, distinct differential requirements were found for MED subunits depending on the promoter. For example, loss of MED13, a subunit of the larger MED complex (ARC-L) thought to play a repressive role in transcription, is not essential for MtnA induction. In contrast, MED13 was found to be important for both MtnB and MtnD activation by MTF-1. In contrast, the opposite specificity was seen with the MED26 subunit, a component of the smaller MED complex (CRSP), thought to play predominantly a coactivator role in transcription. Interestingly, MED26 is required for full induction of the MtnA promoter but is dispensable for MTF-1 activation of the MtnB and MtnD promoters. Thus, these experiments reveal a remarkable example of differential dependence on cofactor composition even though all three promoters tested use the same activator. Apparently, the precise role of individual MED subunits depends on the promoter context and structure, despite the absence of any evidence of direct binding of DNA by the MED complex (Marr, 2006).

To help rule out nonspecific effects on transcription such as a change in the concentration of free RNA polymerase, representative targets from TFIID and MED were tested in a transient transfection assay where the effect to a second promoter can be normalized. In these experiments, TAF4 and MED17 were chosed as representative targets, since TAF4 compromises much of the TFIID complex and MED 17 is likely a component of the core MED complex. The transient transfection data are largely consistent with the data generated at the endogenous locus and at the transgene (Marr, 2006).

The data presented above suggest that activation of the MtnA gene requires specific MED subunits, and at the same time the TAFs appear to be playing a potential negative regulatory role. Because it is clear that the TAFs are specifically recruited in S2 cells to the MtnA promoter in a copper-dependent manner by MTF-1, whether TFIID recruitment can occur in the absence of the MED complex was examined. To achieve this, RNAi directed against MED17 was used, which results in an almost complete loss of MED activity. Surprisingly, TFIID is still efficiently recruited to the MtnA gene. ChIP experiments confirmed that TBP and TAF2 are still actively (and likely directly) recruited to the endogenous MtnA gene by MTF-1 even when the gene is transcriptionally inactive as measured by qPCR analysis. The MtnA luciferase transgene system was used to investigate this relationship at the single-cell level. Without any RNAi, TBP, TAF2, and RNAPII were all recruited to the transgene. In agreement with the ChIP data above, even in the absence of MED activity, after MED17 depletion, TBP and TAF2 are nevertheless efficiently recruited to the transgene. In contrast, no RNAPII can be detected at the transgene consistent with the loss of transcription activation. Apparently, TFIID is recruited to the promoter, but the promoter is not active in supporting transcription. Importantly, recruitment of this 'inactive TFIID' is dependent on the activator MTF-1. In the absence of MTF-1, no TFIID or RNAPII is recruited to the transgene (Marr, 2006).

This perplexing result of recruiting an apparently 'inactive' TFIID prompted an examination of what happens when both TAFs and MEDs were depleted. Remarkably when both the TAFs and MED complex are depleted and 'removed' from the MtnA promoter, MTF-1-dependent activation of transcription is restored to ~95% the level of untreated cells, which is well above the inhibited level observed when the MEDs alone are depleted. In humans and Drosophila, TAFs can be subunits of other complexes such as TFTC and STAGA, so it is possible that the functional interaction analyzed is not TFIID-specific. To test this, specific subunits of these other complexes were targeted to determine if they would have a similar ability to rescue the MED knockdown. Unlike the TFIID subunits, RNAi against dAda2b, dGCN5, dSPT3, and dTRA1 was unable to rescue the loss of the MED subunits. These findings taken together suggest that most likely the functional relationship revealed by these experiments with the MtnA promoter, indeed, involve some regulatory transaction between TFIID and MED (Marr, 2006).

The requirement for coactivator complexes mediating transcriptional responses to activators has been well documented. However, by using an inducible Drosophila gene as a model system, a previously unknown functional interaction has been uncovered between two coactivator complexes, TFIID and MED. In the absence of TAFs, the cell responds inappropriately to a metal stimulus. The cell synthesizes 50%–200% more mRNA from the MtnA gene than it does in the presence of the TAFs. The data suggest that at this gene, TFIID is recruited in an inactive state, a state that impedes initiation of transcription. It is believed that this sets up a checkpoint early in the initiation process to meter the RNA synthesis. The MED complex must be recruited to get past this checkpoint. It is postulated that the MED complex likely modifies TFIID, converting it to an active state. This could be accomplished either through one of the known enzymatic activities of MED, phosphorylating (cdk8) or ubiquitylating (MED8) TFIID subunits, or through some, as yet undetected, chaperone-like function that remodels TFIID into an active conformation. Not surprisingly then, in the absence of MED subunits the cell cannot mount an appropriate response to environmental signals. In fact, depletions of many of the MED subunits lead to <20% of the normal amount of mRNA. Unlike the uniform response to depletion of TAFs, the response to depletion of MEDs is much less uniform. One possibility is that the MED complex is more functionally and structurally diverse than TFIID. Indeed, alternative subcomplexes of MED have been purified biochemically, whereas no such subcomplexes of TFIID have been reported (Marr, 2006).

By analysis of three different Mtn genes, all of which are dependent on the same single activator, it was found, surprisingly, that there is a differential requirement of specific MED subunits at the three Mtn promoters. This is taken as evidence that, depending on the precise arrangement of cis elements and promoter context, the same activator can require different mediator subunits or modules to transmit its signals to the basal apparatus (Marr, 2006).

Interestingly, the kinase module of the MED complex, previously linked with repression functions, is required for efficient activation at two of the promoters. This result, combined with the finding that at the MtnA promoter the TAFs have a repressive regulatory influence on transcription initiation, underscores the difficulty in assigning black and white functions to the coactivator complexes. It is likely that both TFIID and MED interpret multiple inputs from cellular signals and act either positively or negatively depending on the signals received as well as the specific promoter context. As such, the complexes may better be viewed as coregulators since they can play either a positive or negative role in the process of modulating gene expression. For example, only when both TFIID and MED are intact do Drosophila S2 cells produce the appropriate amounts of MtnA mRNA. In contrast, when either coactivator complex is disrupted, aberrant levels of transcription are seen. However, when both coactivator complexes are depleted, a significant level of metal inducible activation is actually restored. Presumably, in this 'stripped down' system, some portion of the remaining TBP pool can mediate transcription. Curiously, in the absence of TAFs but with a full complement of MEDs, there is also an aberrant level of transcription consistent with the notion that there is some finely tuned codependence between the TBP/TAF complex and the MED complex at this promoter (Marr, 2006).

The results also reinforce the notion that the activator is the primary determinant of the transcriptional response. The MTF-1 depletion experiments were the most detrimental to mRNA induction. In the absence of MTF-1, there is no detectable activation of the Mtn genes. In contrast, there is some residual transcription of MtnA even when either the MEDs or TBP are largely depleted from the Drosophila cells. This remaining activity could be due to incomplete depletion, or it could indicate alternative mechanisms of activation that are activator-dependent but can partially bypass the requirement for the coregulator complexes (Marr, 2006).

In the course of testing the requirement for TAFs in activated transcription, the codependent stability of the TFIID complex was discovered. Particularly striking is the finding that TAF4 depletion destabilizes most of the other TAFs and, to some extent, even TBP. Therefore, the TAF depletion experiments most likely reflect a loss of holo-TFIID rather than just the loss of individual subunits. It is worth noting that metazoan organisms contain multiple variants of TAF4: TAF4b in vertebrates and No-hitter in Drosophila. Both of these have been implicated in tissue-specific gene expression. It is conceivable that substitution of this keystone TAF can provide a mechanism to change the entire coregulator profile of TFIID (Marr, 2006).

One intriguing question this work raises is: Why would an activator recruit an inactive TFIID complex to the promoter? There are several previously described cases in which TFIID occupancy at a promoter does not strictly correlate with transcriptional activity. However, in most of these cases the genes being examined were either in a repressed or an unstimulated state. In contrast, the current studies were designed to specifically measure the role of coactivator complexes such as TFIID and MED in the context of an active gene MtnA upon metal stimulation. The ability to deplete MED activity under these conditions revealed the unexpected finding that although TFIID is dynamically recruited to the MtnA promoter, TFIID is mainly held in an 'inactive' state until the second cofactor complex, MED, is recruited. Perhaps this recruitment of an 'inactive' TFIID is a more common phenomenon that can only be detected in special circumstances and may represent a previously unappreciated control mechanism in transcription activation. If the activator first recruits TFIID, then subsequently recruits MED, and there is a requirement for additional factors to potentiate the secondary recruitment of coregulator assemblies, then this provides a potential checkpoint for fine-tuning the control of gene expression. Alternatively, since the cell invests a significant amount of energy in making a high level of transcript, requirement of continued stimulation (i.e., activator bound at the promoter) for mRNA production would provide the most economical use of resources (Marr, 2006).

Targets of Activity

The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals

Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. This study characterizes the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis (Zhang, 2001).

In Drosophila, the two genes encoding MTs, Mto and Mtn, have distinct but partially overlapping expression patterns, with Mto being primarily expressed in early embryogenesis and Mtn being expressed in late embryogenesis/adulthood. In addition, Mtn is expressed in hemocytes, possibly to regulate copper supply to hemocyanin. It was postulated that Drosophila Mto is important for copper homeostasis during embryogenesis, while Mtn, in particular due to its very strong expression in the gut, Malpighian tubules, and fat body, is thought to balance the toxic effects of copper and other metals, such as cadmium and mercury. Thus, Mtn seems to play a role that in the snail is delegated to two different MTs, one active in hemocytes, the other one in the gut (Zhang, 2001 and references therein).

This study reports the isolation of Drosophila dMTF-1. The expression pattern of dMTF-1 is compatible with a role in MT gene activation/heavy metal detoxification and homeostasis. Furthermore, in transfection experiments both in mammalian cells and Drosophila S2 cells, dMTF-1 is able to confer strong cadmium induction to MT promoters of both Drosophila and mammalian origins. As in mammals, metal induction seems to involve characteristic MRE sequence motifs. In support of this notion, the promoters of both Drosophila MT genes, Mto and Mtn, contain multiple sequence motifs resembling mammalian MREs. Their role in metal response was further corroborated by a synthetic minipromoter where the four MREs from the Mto gene were juxtaposed in a tandem array and conferred strong heavy metal response to the reporter gene. In spite of some striking similarities, it should also be pointed out that significant differences exist between dMTF-1 and vertebrate MTF-1. First of all, the strongly conserved zinc finger domain notwithstanding, there is limited, albeit significant, protein sequence similarity between vertebrate MTF-1 and dMTF-1. Specifically, the three hallmark activation domains of mammalian MTF-1, namely, acidic, proline-rich, and serine/threonine-rich, do not have obvious counterparts in Drosophila. The functional equivalents of one or all of them remain to be identified. Secondly, dMTF-1 is quite forgiving towards low pH (6.0 to 6.5) while mammalian MTF-1 loses its DNA-binding capacity under these conditions. This property may explain why mammalian MTF-1 was inactive in transfected Drosophila Schneider cells, which are grown at pH 6.5. Conversely, dMTF-1 performed well in mammalian cells grown in Dulbecco modified Eagle medium at pH 7.4. A third difference may concern the tissue distribution: while mammalian MTF-1 is expressed in all tissues analyzed so far, dMTF-1 is expressed in a few tissues, notably gut and fat body, although further experiments will have to probe into this issue. This is compatible with a major role in the activation of MT and possibly other toxicity/cell stress-related genes. Finally, there is a difference between mammals and Drosophila concerning heavy metal metabolism. In mammalian cells, MTF-1 mediates MT gene transcription primarily in response to zinc and cadmium and, to a lesser extent, to copper and nickel. Surprisingly dMTF-1 was unable to induce transcription in response to zinc at concentrations which readily induce transcription in mammalian cells. However, at 2 mM zinc, a concentration that is lethal to mammalian cells, dMTF-1 activated transcription from the Mtn promoter. This finding is in agreement with earlier heavy metal studies of the fly: of all metals tested for Mto and Mtn transcription, Cd2+, Cu2+, and Hg2+ were found to be more efficient than Zn2+, which was required at concentrations of at least 1 mM. The high concentrations of zinc (and copper) tolerated by Schneider cells may reflect inefficient uptake or, more likely, particularly efficient export of these two essential heavy metals. In fact, a search of the Drosophila genome revealed six homologs of mammalian zinc transporters. Whatever the reason for the poor response to zinc at submillimolar concentrations, it cannot be attributed to a species difference between MTF-1 factors themselves, since dMTF-1, upon transfection into mouse cells, mediates zinc-responsive transcription like hMTF-1. Further evidence for functional similarity is that dMTF-1, like mammalian MTF-1, requires elevated zinc concentrations for DNA binding in vitro, while copper and cadmium interfere with zinc rather than replacing it. At first sight, cadmium and copper induction of a transcription factor that strictly requires zinc appears paradoxical. However, it is possible to induce MTF-1-dependent transcription in vitro by cadmium and copper. Activation is dependent on the presence of MT, which has a particularly high affinity for these two metals and upon binding to them releases zinc on behalf of MTF-1. Another question concerns the regulation of the dMTF-1 gene itself. In mammals, the MTF-1 promoter does not contain MREs and transcripts are marginally, if at all, elevated by heavy metal treatment. Although MTF-1 transcripts have not been quantitated in Drosophila, it is noted that the MTF-1 promoter, like the mammalian one, lacks MRE-type sequence motifs, at least within a 2-kb segment around the site of transcription initiation (Zhang, 2001).

Perhaps the best evidence for an important role of dMTF-1 in MT gene regulation is provided by studies with RNAi. Without RNAi and in the absence of any Drosophila mutation in the dMTF-1 gene, one might have argued that the observed effect in transfected cells was due to a nonphysiological stimulation by a heterologous zinc finger factor. Ectopic activation effects have been observed before, when related family members of mammalian transcription factors could replace each other in transient transfection/overexpression conditions. This scenario is ruled out by the finding that dMTF-1 dsRNA completely blocked the activation of the Mtn promoter without any cotransfected MTF-1 expression plasmid, i.e., under conditions that relied entirely on the host cell's transcription factors. Furthermore, unlike many other transcription factors, MTF-1 is a unique protein without related family members in Drosophila and, apparently, in mammals. Taken together, the RNAi experiments corroborated the important role of dMTF-1 for MT transcription and thus heavy metal homeostasis (Zhang, 2001).

It certainly will be of interest to study the effect of loss of dMTF-1 in vivo, for example, by screening deletion mutants at the MTF-1 locus or by the newly introduced techniques of inheritable RNAi or targeted gene disruption in Drosophila. As mentioned, a targeted disruption of MTF-1 in the mouse results in embryonic lethality due to liver decay on embryonic days 13 to 14. It is noted that disruption of either of two other stress-associated transcription factors, namely, c-Jun and NF-kappaB/RelA, also results in embryonic death from liver decay at about the same stage of mouse embryogenesis. In this context it is of interest that AP-1 sites, which bind jun-fos heterodimers, are present in the promoters of both Drosophila MTs, pointing to a possible interconnection of MTF-1 and AP-1 in the cellular stress response of insects. Thanks to the availability of jun and fos, mutants and the power of Drosophila genetics, these and other aspects of cellular stress response are amenable to analysis (Zhang, 2001).

Metal-responsive transcription factor (MTF-1) and heavy metal stress response in Drosophila and mammalian cells: A functional comparison

The zinc finger transcription factor MTF-1 is conserved from insects to vertebrates. Its major role in both organisms is to control the transcription of genes involved in the homeostasis and detoxification of heavy metal ions such as Cu2+, Zn2+ and Cd2+. In mammals, MTF-1 serves at least two additional roles. First, targeted disruption of the MTF-1 gene results in death at embryonic day 14 due to liver degeneration, revealing a stage-specific developmental role. Second, under hypoxic-anoxic stress, MTF-1 helps to activate the transcription of the gene placental growth factor (PIGF), an angiogenic protein. dMTF-1, the Drosophila homolog of mammalian MTF-1, has been cloned and characterized. This study presents a series of studies to compare the metal response in mammals and insects, that reveal common features but also differences. A human MTF-1 transgene can restore to a large extent metal tolerance to flies lacking their own MTF-1 gene, both at low and high copper concentrations. Likewise, Drosophila MTF-1 can substitute for human MTF-1 in mammalian cell culture, although both the basal and the metal-induced transcript levels are lower. Finally, a clear difference was revealed in the response to mercury, a highly toxic heavy metal: metallothionein-type promoters respond poorly, if at all, to Hg2+ in mammalian cells but strongly in Drosophila, and this response is completely dependent on dMTF-1 (Balamurugan, 2004).

Protein Interactions

Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans (Hsu, 1994: Hsu, 1995). Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. These studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility (Vardanyan, 2008).

In transfected cells, both of the Dpy-30 orthologs of Drosophila, termed Dpy-30L1 and Dpy-30L2 (Dumpy-30-like1 and Dumpy-30-like2), inhibit the activity of MTF-1 (metal-responsive transcription factor 1), while in transgenic flies, such an effect was only seen with the stronger interactor Dpy-30L1. Consistent with such an inhibition, transgenic flies were sensitive to cadmium or zinc load, while copper sensitivity was only marginally affected. The increased metal sensitivity could be rescued by co-overexpression of dMTF-1. An EMSA assay revealed a weakened binding of MTF-1 to MRE DNA in the presence of Dpy-30L1. Taken together, these results suggest that for detoxification of Cd(II) or Zn(II) a higher level of MTF-1 is required than for Cu(II) detoxification. Studies with partial inactivation mutants of dMTF-1 are in agreement with such a notion. Unexpectedly, only MTF-1 of insect origin responded to Dpy-30 type proteins: while the human and mouse Dpy-30 members also inhibited Drosophila MTF-1 across species boundaries, activity of human MTF-1 was unchanged in the presence of either Drosophila or mammalian Dpy-30 members. This indicates some degree of functional divergence between Drosophila and mammalian MTF-1 during evolution, in spite of a conserved role in heavy metal homeostasis and detoxification. The Dpy-30-dMTF-1 interactions observed in the interactome study are considered relevant because (1) the two major interactors Dpy-30L1 and L2 are members of the same protein family; (2) a (negative) functional interaction with dMTF-1 was seen with both of them, and with their mammalian Dpy-30 homolog, in transfected cells; (3) Dpy-30L1, the stronger interactor, also produced an effect in vivo, and (4) it inhibited the binding of dMTF-1 to its cognate DNA sequence (Vardanyan, 2008).

As a complement to transgenic expression of Dpy-30L1 and Dpy-30L2, loss of function of the two proteins was tested. Disruption of short genes in Drosophila has been a great challenge since small targets are rarely hit by random mutagenesis. To circumvent this problem, Dpy-30L1 and L2 function were eliminated separately by homologous recombination. Somewhat unexpectedly, knockout of neither Dpy-30L1 nor Dpy-30L2 affected metal handling under the conditions tested, but Dpy-30L2 which is specifically expressed in male gonads, turned out to be essential for male fertility (Vardanyan, 2008).

Sdc1, the yeast homolog of Dpy-30, is a component of SET1C, also called COMPASS (complex proteins associated with SET1 protein (Nagy, 2002). SET1C methylates histone H3 at lysine residue 4. Yeast strains mutant for SET1, although viable, display defects in cell growth, rDNA silencing (Briggs, 2001), and silencing of telomeres and mating type loci (Nislow, 1997). In C. elegans, the dosage compensation complex (DCC), which among other proteins includes Dpy-30, represses X-chromosomal transcription in cells of XX animals. The complex binds preferentially to promoter regions and seems to be required for the early steps of dosage compensation, not for its maintenance (Ercan, 2007). The SET1C complex has also been shown to activate some specific genes, notably for DNA repair genes. This activation is however an indirect one, via repression of a signaling cascade. Direct activation of target genes is also possible, at least in mammals: a human homolog of SET1C, the MLL (mixed-lineage leukemia) complex which also has methyltransferase activity and is ivolved in tumor cell proliferation (Milne, 2002), positively regulates Hox gene expression through binding to promoter sequences. Recent investigations have shown that the human MLL2/ALR complex contains the human ortholog of Dpy-30. Taken together, these data indicate a conserved role of Dpy-30 family members in the modulation of chromatin structure and transcription (Vardanyan, 2008).

However, there are clear differences as well. The Drosophila trithorax complex, the homolog of yeast SET1C, is essential for viability. The current findings suggest that flies lacking both Dpy-30L1 and Dpy-30L2 are viable and that Dpy-30 orthologs of Drosophila are not obligatory components of the trithorax complex. The only mutant phenotype observed was male sterility in the absence of Dpy-30L2. A hallmark of spermatogenesis, the replacement of histones by protamines is not affected in the Dpy-30L2 mutant. Because transcriptional silencing of the spermatid genome seems to occur independently of protamines, it appears still possible that Dpy-30L2 is required for proper gene silencing during spermatogenesis (Vardanyan, 2008).

In yeast, C. elegans and Drosophila, Dpy-30 members serve different but important functions, perhaps converging, in metazoans, on sex-specific gene expression programs, compatible with the fact that the single Dpy-30 ortholog of the mouse is strongly expressed in testes (Vardanyan, 2008).

In conclusion, Dumpy-30 (Dpy-30) type proteins are conserved from yeast to humans but their function in higher eukaryotes is only partially understood. This study has characterized the two Dpy-30 familiy members in Drosophila. Strong expression of Dpy-30L1 can inhibit the activity of MTF-1 (metal-responsive transcription factor 1), resulting in elevated sensitivity of flies to cadmium and zinc load. The second member, Dpy-30L2, is only expressed in the male genital tract; targeted gene disruption of dpy-30L2 results in male sterility associated with reduced motility of sperm and failure to be transferred to the female's seminal receptacles. Like Drosophila Dpy-30L2, the mouse Dpy-30 homolog is strongly expressed in testes, from where the expressed sequence tag (EST) was obtained. Thus Dpy-30 family members may well be required for male fertility also in mammals (Vardanyan, 2008).


DEVELOPMENTAL BIOLOGY

A Northern blot was performed with samples of Drosophila MTF-1 poly(A) RNA; a steady increase of dMTF-1 mRNA was detected in embryos, larvae, and pupae relative to the mRNA for ribosomal protein L32. In order to obtain some information on the expression of dMTF-1 in the adult, frozen tissue sections were subjected to in situ hybridization. Preliminary results indicate a strong expression in the fat body and in the gut, consistent with a major role in the control of MT (Mtn), which is expressed in the gut and also the fat body of the adult fly (Zhang, 2001).


EFFECTS OF MUTATION
'Metal-responsive transcription factor-1' (MTF-1), a zinc finger protein, is conserved from mammals to insects. In the mouse, it activates metallothionein genes and other target genes in response to several cell stress conditions, notably heavy metal load. The knockout of MTF-1 in the mouse has an embryonic lethal phenotype accompanied by liver degeneration. This study describes the targeted disruption of the MTF-1 gene in Drosophila by homologous recombination. Unlike the situation in the mouse, knockout of MTF-1 in Drosophila is not lethal. Flies survive well under laboratory conditions but are sensitive to elevated concentrations of copper, cadmium and zinc. Basal and metal-induced expression of Drosophila metallothionein genes MtnA (Mtn) and MtnB (Mto), and of two new metallothionein genes described in this study, MtnC and MtnD, is abolished in MTF-1 mutants. Unexpectedly, MTF-1 mutant larvae are sensitive not only to copper load but also to copper depletion. In MTF-1 mutants, copper depletion prevents metamorphosis and dramatically extends larval development/lifespan from normally 4-5 days to as many as 32 days, possibly reflecting the effects of impaired oxygen metabolism. These findings expand the roles of MTF-1 in the control of heavy metal homeostasis (Egli, 2003).

Every organism must cope with environmental fluctuation of heavy metal concentrations. Non-essential, toxic heavy metals have to be exported or sequestered intracellularly, while the uptake, storage and distribution of essential heavy metals, such as zinc and copper, have to be ensured, with the additional problem that even these metals are toxic if present in excess (Egli, 2003).

Important components of the heavy metal homeostasis and detoxification system are the membrane-based heavy metal transporters, intracellular metal chaperones for efficient distribution of scarce essential metals, and the metallothioneins, a group of small, cysteine-rich proteins that have the ability to bind and thereby sequester heavy metals. There are >10 functional metallothionein genes in humans and four in the mouse; in Drosophila, two genes were characterized before, designated Mtn/MtnA and Mto/MtnB. Transcription of metallothionein genes is strongly induced by heavy metal load. This induction is mediated via conserved DNA sequence motifs, so-called metal response elements (MREs) of consensus TGCRCNC (R = A or G, and N = any nucleotide) that are present in the promoters of all metallothionein genes (Stuart, 1985) from insects to mammals (Egli, 2003).

A zinc finger transcription factor has been characterized that binds to MRE sequences. This protein was referred to as metal response element-binding transcription factor-1 (MTF-1, also designated metal-responsive transcription factor-1 or metal transcription factor-1) (Westin, 1988; Radtke, 1993; Brugnera, 1994; Auf der Maur, 1999; reviewed in Andrews, 2001; Giedroc, 2001; Lichtlen, 2001). In the mouse, MTF-1 plays an essential role in liver development; targeted deletion of the MTF-1 gene results in embryonic death due to liver degeneration (Günes, 1998). A search for MTF-1 target genes in the mouse has revealed metallothionein genes and a number of other genes, several of which contain MREs in the promoter and/or are involved in coping with cell stress (Andrews, 2001; Lichtlen, 2001). MTF-1 plays a role not only in heavy metal stress but also in other cell stress conditions (Murphy, 1999; Dalton, 2000; Adilakshmi, 2002; reviewed in Lichtlen, 2001) such as oxidative stress, hypoxia and amino acid starvation (Egli, 2003).

The MTF-1 homolog from Drosophila can activate metallothionein gene promoters in cell transfection experiments (Zhang, 2001). Inactivating the Drosophila MTF-1 gene using targeted gene disruption by homologous recombinationyields viable flies that are sensitive not only to high concentrations of copper, cadmium and zinc but, unexpectedly, also to copper depletion. Also, two novel metallothionein genes MtnC and MtnD are described and shown to be, like the known metallothionein genes MtnA and MtnB, targets of MTF-1 (Egli, 2003).

Unlike the MTF-1 mutant mice, which die from embryonic liver degeneration in utero, MTF-1 mutant flies are viable yet sensitive to heavy metals and copper depletion. This phenotype is very similar for combinations of a variety of different alleles including an initiator triplet mutation, a 1.1 kb deletion, a 4.1 kb deletion, and tandem or single mutant genes in combination with a chromosomal deficiency. Subtle differences exist among different allelic combinations in their sensitivity to either heavy metal load or copper chelators. The allele MTF-1140-1R carrying a 4.1 kb deletion of the coding region possesses the strongest phenotype and is most probably a null mutation (Egli, 2003).

In general, MTF-1 mutants are sensitive to distortions of heavy metal balance. One aspect, namely the sensitivity to high concentrations of cadmium but also to copper and zinc, is in agreement with earlier findings with cultured cells of MTF-1-/- mice (Günes, 1998) and Drosophila (Zhang, 2001). The failure of MTF-1 mutants to induce metallothionein genes provides the most likely explanation for their sensitivity to heavy metal load (Egli, 2003).

Quite unexpected, however, is the exquisite sensitivity of MTF-1 mutants to copper depletion. This is particularly interesting because scarcity of trace heavy metals is probably encountered more often under natural conditions than heavy metal load. It is worth mentioning that another Drosophila mutant displays a similar phenotype: flies with a deletion of a copper transporter (Ctr1B) are also sensitive to both excess copper and copper depletion (D. J. Thiele, personal communication to Egli, 2003). The mechanism for this dual sensitivity may be a translocation of the protein from the outer membrane to vacuoles/lysosomes under limiting and excess copper concentrations, respectively. So far, it is unclear how MTF-1 enables a cell to cope with metal depletion. The results suggest that MTF-1 regulates either import or efficient usage of copper, since wild-type flies may be grown continuously on copper chelator food, whereas MTF-1 mutants are able to do so under sublethal conditions for just a single generation. It is speculated that MTF-1, upon copper depletion, activates a copper import pump and/or inactivates an export pump or, alternatively, regulates the expression of a copper chaperone. Drosophila metallothioneins themselves, which preferentially bind copper (Valls, 2000), may act not only as heavy metal scavengers upon heavy metal load, but also, under limiting copper concentrations, as copper chaperones similar to Cox17 and Atx1 (reviewed in Harrison, 1999). Alternatively, Drosophila copper-loaded metallothioneins may act as a storage pool for copper (see also Dalton, 1996). This scenario is compatible with the finding that MTF-1 is also required for the basal transcription of metallothionein genes. Thus, the lack of metallothioneins in MTF-1 mutants may also be responsible for their sensitivity to copper depletion (Egli, 2003).

Another enigma is the extreme extension of larval development in the presence of copper chelator in the food. It is not clear at present whether this prolonged larval period involves a genuine longevity effect. Several hypotheses can be envisaged to explain this phenomenon. (1) Extension of larval life could be due to a decrease in cytochrome c oxidase (COX) activity, a key copper-containing enzyme in the respiratory chain. In the fungus Podospora anserina, elimination of grisea, a copper-modulated transcription factor, affects the expression of a copper transporter and leads to impaired copper uptake. This correlates with a reduced activity of the COX complex and is associated with delayed growth and an extended lifespan. The same phenotype is produced by the direct elimination of COX5, a subunit of the cytochrome c oxidase. Extension of lifespan in P.anserina can also be achieved by the mere addition of the copper chelator BCS to the medium. In the case of Drosophila, a decrease in cytochrome c oxidase activity due to insufficient copper supply could reduce ATP production, slow growth and, consequently, prolong the larval period. (2) Copper in contrast is an essential component of enzymes, including tyrosinase/phenol oxidase and superoxide dismutase for radical scavenging, but in contrast contributes directly to the formation of oxygen radicals in the Fenton reaction; for the latter reason, copper depletion may result in less oxidative damage. This by itself would not explain stalled development, but rather why larvae survive that long. (3) Mutant larvae raised with a copper chelator grow but retain features of second instar larvae including thinner tracheal ducts; thus insufficient oxygen supply could restrict growth and prolong the larval period, perhaps again in combination with less oxidative damage of tissues. (4) Larvae raised in chelator-containing food also have smaller mouth hooks, which may prevent them from using the food efficiently. Thus extended larval life could be the result of a caloric restriction, which is known to delay growth and extend lifespan in a large variety of organisms from yeast to mammals. This is considered unlikely, because starvation due to a 10-fold dilution of the food cake does not reveal any differences between wild-type and MTF-1 mutant larvae. (5) The gene for SHC adaptor protein (shc) involved in tyrosine kinase receptor signaling is located quite close to the MTF-1 transcription unit (two genes upstream, at 3.3 kb distance), and in the mouse, knockout of shc has been found to extend lifespan. Thus the knockout of MTF-1 might adversely affect regulatory sequences of the shc gene. However, the simple mutation of three bases at the MTF-1 translation initiation codon in Drosophila is unlikely to have such an effect. Further more, sequencing of the genomic region in the mutant flies revealed no difference from wild-type in the shc or Rps17 region, showing that the targeting process has not affected these two neighboring genes (Egli, 2003).

Whatever the reason for this greatly expanded larval period, the results firmly establish for MTF-1 a central role in the heavy metal metabolism of the fly. The exquisite sensitivity of MTF-1 mutants to copper depletion points to a new role for this protein that waits to be explored also in mammals (Egli, 2003).


EVOLUTIONARY HOMOLOGS

Cloning and characterization of MTF-1

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. The presence of an MRE-binding factor has been demonstrated in HeLa cell nuclear extracts. This factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. The amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. The cDNA of mouse MTF-1, a 72.5 kDa protein, was cloned. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels (Radtke, 1993).

Metallothioneins (MTs) are small cysteine-rich proteins that bind heavy metal ions such as zinc, cadmium and copper with high affinity, and have been functionally implicated in heavy metal detoxification and radical scavenging. Transcription of metallothioneins genes is induced by exposure of cells to heavy metals. This induction is mediated by metal-responsive promoter elements (MREs). The cDNA of an MRE-binding transcription factor (MTF-1) from the mouse has been cloned. This study presents the human cDNA equivalent of this metal-regulatory factor. Human MTF-1 is a protein of 753 amino acids with 93% amino acid sequence identity to mouse MTF-1 and has an extension of 78 amino acids at the C-terminus without counterpart in the mouse. The factors of both species have the same overall structure including six zinc fingers in the DNA binding domain. The human MTF-1 gene has been mapped to human chromosome 1 where it localizes to the short arm in the region 1p32-34, most likely 1p33. Both human and mouse MTF-1 when produced in transfected mammalian cells strongly bind to a consensus MRE of metallothionein promoters. However, human MTF-1 is more effective than the mouse MTF-1 clone in mediating zinc-induced transcription (Brugnera, 1994).

The pufferfish Fugu rubripes contains a full set of vertebrate genes but only 13% as much DNA as a mammal. Fugu genes tend to be smaller and densely spaced due to shortening of introns and intergenic spacers. The Fugu gene for the metal-responsive transcription factor MTF-1 (MTF1), a mediator of heavy metal regulation and oxidative stress response previously characterized in mammals, was isolated. In addition, most of the cDNA sequence was also determined. The 780 amino acid MTF-1 protein of Fugu is very similar to that of mouse and human, with 90% amino acid identity in the DNA binding zinc finger domain and 57% overall identity. Expression of the pufferfish cDNA in mammalian cells shows that Fugu MTF-1 has the same DNA binding specificity as its mammalian counterpart and also induces transcription in response to zinc and cadmium. The protein-coding part of the Fugu MTF-1 gene spans 6.4 kb and consists of 11 exons. Upstream region and first exon constitute a CpG island. The distance between stop codon and polyadenylation motifs is >2 kb, suggesting a very long 3' untranslated mRNA region, followed by another CpG island which may represent the promoter of the next gene downstream. Part of the MTF-1 genomic structure was also determined in the mouse, and some striking similarities were found: for example, the upstream adjacent gene in both species is INPP5P, encoding a phosphatase. The mouse MTF-1 promoter is also embedded in a CpG island, which however shares no sequence similarity to the one of Fugu. The Fugu CpG island is shorter than the one of the mouse and has no elevated [G+C] content; these and other data indicate that CpG islands of fish may represent a primordial stage of CpG island evolution (Auf der Maur, 1999).

The metal response element (MRE)-binding transcription factor-1, MTF-1, is a zinc-responsive protein that controls transcription of metallothionein (MT) genes in many cell types. In addition, MTF-1 is also hypothesized to regulate transcription of a battery of genes involved in the defense against oxidative stress. Manipulating the Zn concentration in the low microM range reversibly modulates the DNA-binding activity of the mammalian MTF-1; this effect is inhibited at low temperature. This report examines the presence and binding properties of MTF-1 in cell lines derived from warm- and cold-water fishes (zebrafish and trout, respectively). Both species of fish express MRE-specific binding activities that are immunologically similar to mouse MTF-1. MTF-1-binding from the cells of both species of fish was activated when cells were treated with Zn but not with Cd. Zebrafish cells contained a single isoform of MTF-1 with binding properties similar to mammalian MTF-1. Trout cells, in contrast, contained two isoforms of MTF-1: MTF-1H and MTF-1L. Zn reversibly modulates MTF-1H binding in a temperature-dependent manner. Similarly, Zn reversibly modulates MTF-1L binding, but, in contrast, such modulation occurs readily at 4 degrees C. This data demonstrates the conservation of binding specificity, binding properties, and regulation of MTF-1 in fishes (Dalton, 2000).

Structure of MTF-1

The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2)-His(2) motifs, each of which was projected to form well-structured betabetaalpha domains upon Zn(II) binding. This study investigated the structure and backbone dynamics of a fragment containing the unusual C-terminal fingers F4-F6. (15)N heteronuclear single quantum coherence (HSQC) spectra of uniformly (15)N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn(3) hMTF-zf46 determined by (13)Calpha chemical shift indexing and the magnitude of (3)J(Halpha-HN) clearly reveal that zinc fingers F4 and F6 adopt typical betabetaalpha structures. An analysis of the heteronuclear backbone (15)N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-MTF-zf46 with Zn(II) reveals that the F4 domain binds Zn(II) significantly more tightly than do the other two finger domains. In contrast to fingers F4 and F6, the ßßalpha fold of finger F5 is unstable and only partially populated at substoichiometric Zn(II); a slight molar excess of zinc results in severe conformational exchange broadening of all F5 NH cross-peaks. Finally, although Cd(II) binds to apo-hMTF-zf46 as revealed by intense S(-)-->Cd(II) absorption, a non-native structure results; addition of stoichiometric Zn(II) to the Cd(II) complex results in quantitative refolding of the betabetaalpha structure in F4 and F6. The functional implications of these results are discussed (Giedrock 2001a).

Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys(2)-His(2) zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d(5)-N-ethylmaleimide (d(5)-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn(6) MTF-zf. A chase with excess H(5)-N-ethylmaleimide (H(5)-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d(5),d(5)-, d(5),H(5)-, and H(5),H(5)-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d(5)-NEM pulse time. This experiment establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 >> F1 > F2 approximately F3 approximately F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that determined for the DNA binding domain of Sp1, Zn(3) Sp1-zf, under identical solution conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-containing oligonucleotide. An identical experiment carried out with Zn(5) MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1 (Apuy, 2001).

Metal-responsive transcription factor 1 (MTF-1) specifically binds to metal response elements (MREs) associated with a number of metal- and stress-responsive genes. Human MTF-1 contains a cysteine-rich cluster, -632Cys-Gln-Cys-Gln-Cys-Ala-Cys638-, conserved from pufferfish to humans far removed from the MRE-binding zinc finger domain and just C-terminal to a previously mapped serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys-->Ala substitutions (C632A/C634A) or a deletion in this region altogether (Delta(632-644)) are significantly impaired in their ability to induce Zn(II)- and Cd(II)-responsive transcription of a MRE-linked reporter gene in transiently transfected mouse dko7 (MTF-1-/-) cells in culture under moderate metal stress but retain the ability to drive basal levels of transcription in a MRE-dependent manner in vivo and in vitro. In addition, the mutated proteins respond to induction by Zn(II) or Cd(II) with nuclear translocation and MRE binding activities comparable with wild-type MTF-1. Attempts to rescue the Delta(632-644) deletion mutant phenotype by inserting similar Cys-rich sequences from Drosophila MTF-1 were unsuccessful, suggesting that the structure of this motif within intact human MTF-1, rather than the simple presence of multiple closely spaced Cys residues, is required for function. This cysteine cluster therefore functions at a step subsequent to nuclear translocation and MRE-binding DNA to naked promoter-containing DNA and appears to be specifically required for MTF-1 to activate transcription in the presence of inducing heavy metal ions (Chen, 2004).

Six Cys(2)His(2) zinc fingers (F1-6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1-6 is necessary for basal and zinc-induced expression of metallothionein genes. Analysis of NMR structural and dynamic data for an F1-6 protein construct demonstrates that each zinc finger adopts a stable ßßalpha fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1-4) and two C-terminal low DNA affinity fingers (F5-6) reveal similar stable zinc finger structures. In both the F1-6 and F5-6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral density and hydrodynamic analysis of (15)N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1-6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys(2)His(2) zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Analysis of NMR (1)H-(15)N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within approximately 10-50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concentration (Potter, 2005).

Mouse metal response element-binding transcription factor-1 (MTF-1) regulates the transcription of genes in response to a variety of stimuli, including exposure to zinc or cadmium, hypoxia, and oxidative stress. Each of these stresses may increase labile cellular zinc, leading to nuclear translocation, DNA binding, and transcriptional activation of metallothionein genes (MT genes) by MTF-1. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF-1 also functions as a zinc-sensing domain. In this study, the potential role of the peptide linkers connecting the four N-terminal zinc fingers of MTF-1 were investigated in their zinc-sensing function. Each of these three linkers is unique, completely conserved among all known vertebrate MTF-1 orthologs, and different from the canonical Cys2His2 zinc finger TGEKP linker sequence. Replacing the RGEYT linker between zinc fingers 1 and 2 with TGEKP abolishes the zinc-sensing function of MTF-1, resulting in constitutive DNA binding, nuclear translocation, and transcriptional activation of the MT-I gene. In contrast, swapping the TKEKP linker between fingers 2 and 3 with TGEKP has little effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorter TGKT linker between fingers 3 and 4 renders MTF-1 less sensitive to zinc-dependent activation both in vivo and in vitro. These observations suggest a mechanism by which physiological concentrations of accessible cellular zinc affect MTF-1 activity. Zinc may modulate highly specific, linker-mediated zinc finger interactions in MTF-1, thus affecting its zinc- and DNA-binding activities, resulting in translocation to the nucleus and binding to the MT-I gene promoter (Li, 2006).

MTF-1 mutants

MTF-1 binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. This study reports the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, evidence is presented that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment (Heuchel, 1994).

The heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, null mutant mice were generated by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. It is also noted that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA) (Günes, 1998).

Metal-responsive transcription factor-1 (MTF-1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryogenesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Mtf1 knockout mice were studied at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild-type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild-type rat embryo hepatocytes, indicating a cell-autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non-stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF-1 in coping with heavy metal stress. An additional MTF-1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF-1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis (Wang, 2004).

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of gamma-ray-induced thymic lymphomas, linkage analysis and haplotype mapping were performed that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development (Tamura, 2005).

Transcriptional targets of MTF-1

Metallothionein genes are transcriptionally regulated by a number of inducers including heavy metals. Previous mutational analyses of the mouse metallothionein-I gene (mMTI) promoter have delineated a heavy-metal regulatory region between -60 and -42 relative to the transcription start site. A synthetic copy of a 12-base-pair (bp) conserved sequence located within this region confers heavy-metal regulation on a heterologous gene. However, specific disruption of this metal regulatory element (MRE) within a wild-type mMTI promoter reduces but does not eliminate the heavy-metal response. The additional metal regulatory activity is localized to an upstream region containing four sequences homologous to the identified MRE. Similar sequences are also found in multiple copies in metallothionein genes from other species. Synthetic copies of all five mMTI MRE homologues were tested for metal regulatory activity. At least four of these sequences are able to confer heavy-metal regulation on a heterologous promoter (Stuart, 1985).

Heavy metal ions are effective inducers of metallothionein gene transcription. The metal response is dependent on short DNA motifs, so-called MREs (metal responsive elements) that occur in multiple copies in the promoter region of these genes. This study analysed an MRE of the mouse metallothionein-I gene (MREd); this can function over long distances as a bona fide metal ion-inducible enhancer. The transcription factor Sp1 and a zinc-inducible factor, designated MTF-1, bind to the MREd enhancer in vitro. The combined use of MREd mutants in a transient assay in HeLa cells and a competition band shift assay show that the zinc-inducible formation of the MTF-1/DNA complex in vitro correlates with zinc-inducible transcription in vivo. A chemical methylation interference assay revealed remarkably similar but non-identical guanine interference patterns for the MTF-1 and Sp1 complexes, which may mean that MTF-1 is related to the Sp1 factor (Westin, 1988).

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. The human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicate that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrate that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d confer hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1-/-; dko7 cells) are not induced by zinc and show low responsiveness to hypoxia. A transiently transfected MT promoter is unresponsive to hypoxia or zinc in dko7 cells, but inductions are restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detect a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1 (Murphy, 1999).

Metal regulation of the mouse zinc transporter (ZnT)-1 gene was examined in cultured cells and in the developing conceptus. Zinc or cadmium treatment of cell lines rapidly (3 h) and dramatically (about 12-fold) induced ZnT1 mRNA levels. In cells incubated in medium supplemented with Chelex-treated fetal bovine serum, to remove metal ions, levels of ZnT1 mRNA are reduced, and induction of this message in response to zinc or cadmium is accentuated (up to 31-fold induction). Changes in ZnT1 gene expression in these experiments parallel those of metallothionein I (MT-I). Inhibition of RNA synthesis blocks metal induction of ZnT1 and MT-I mRNAs, whereas inhibition of protein synthesis does not. Metal response element-binding transcription factor (MTF)-1 mediates metal regulation of the metallothionein I gene. In vitro DNA-binding assays demonstrate that mouse MTF-1 can bind avidly to the two metal-response element sequences found in the ZnT1 promoter. Using mouse embryo fibroblasts with homozygous deletions of the MTF-1 gene, it was shown that this transcription factor is essential for basal as well as metal (zinc and cadmium) regulation of the ZnT1 gene in these cells. In vivo, ZnT1 mRNA is abundant in the midgestation visceral yolk sac and placenta. Dietary zinc deficiency during pregnancy down-regulates ZnT1 and MT-I mRNA levels (4-5-fold and >20-fold, respectively) in the visceral yolk sac, but has little effect on these mRNAs in the placenta. Homozygous knockout of the MTF-1 gene in transgenic mice also leads to a 4-6-fold reduction in ZnT1 mRNA levels and a loss of MT-I mRNA in the visceral yolk sac. These results suggest that MTF-1 mediates the response to metal ions of both the ZnT1 and the MT-I genes the visceral yolk sac. Overall, these studies suggest that MTF-1 directly coordinates the regulation of genes involved in zinc homeostasis and protection against metal toxicity (Langmade, 2000).

The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins that participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents. In mice, among the four known MT genes, the MT-I and -II genes are most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger metal-responsive transcription factor MTF-1 plays a central role in transcriptional activation of the MT-I gene in response to metals and oxidative stress. Mutation of the MTF-1 gene abolishes these responses, and MTF-1 is induced to bind to the metal response elements in proximal MT promoter in cells treated with zinc or during oxidative stress. The exact molecular mechanisms of action of MTF-1 are not fully understood. These studies suggest that the DNA-binding activity of MTF-1 in vivo and in vitro is reversibly activated by zinc interactions with the zinc-finger domain. This reflects heterogeneity in the structure and function of the six zinc fingers. It is hypothesized that MTF-1 functions as a sensor of free zinc pools in the cell. Changes in free zinc may occur in response to chemically diverse inducers. MTF-1 also exerts effects on MT-I gene transcription which are independent of a large increase in MTF-1 DNA-binding activity. For example, cadmium, which has little effect on the DNA-binding activity of MTF-1 in vivo or in vitro, is a more potent inducer of MT gene expression than is zinc. The basic helix-loop-helix-leucine zipper protein, USF (upstream stimulatory factor family), also plays a role in regulating transcription of the mouse MT-I gene in response to cadmium or H2O2. Expression of dominant negative USF-1 or deletion of its binding site from the proximal promoter attenuates induction of the mouse MT-I gene. USF apparently functions in this context by interacting with as yet unidentified proteins which bind to an antioxidant response element which overlaps the USF-binding site (USF/ARE). Interestingly, this composite element does not participate in the induction of MT-I gene transcription by zinc or redox-cycling quinones. Thus, regulation of the mouse MT-I gene by metals and oxidative stress involves multiple signaling pathways which depend on the species of metal ion and the nature of the oxidative stress (Andrews, 2001).

Coordinate regulation of the ribosomal protein genes is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes. A cellular model system has been uncovered that selectively induces the ribosomal protein S25 gene in hepatoma cells that are stressed by nutrient deprivation. The results indicate that p53 along with two other identified proteins, MTF-1 and La, post-transcriptionally regulate the synthesis of the S25 protein by controlling the nuclear export of the stress-induced S25 mRNA. This system is unique in that the nuclear-retained S25 mRNA is exported to the cytosol only upon replenishment or alternatively after prolonged starvation to participate in a p53-mediated apoptotic sequence of events. This p53-dependent survival or death pathway involves a previously unreported protein relationship among these three actors, one of which, MTF-1, has not yet been shown to have RNA-binding characteristics (Adilakshmi, 2002).

Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein W (Wimmer, 2005).

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. PlGF expression is also controlled by NF-kappaB. Several putative binding sites for NF-kappaB were identifed in the PlGF promoter/enhancer region by sequence analyses, and binding and transcriptional activity of NF-kappaB p65 was demonstrated at these sites. Expression of NF-kappaB p65 from a plasmid vector in HEK293 cells causes a substantial increase of PlGF transcript levels. Furthermore, hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-kappaB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlGF (Cramer, 2006).


REFERENCES

Search PubMed for articles about Drosophila Metal response element-binding Transcription Factor-1

Adilakshmi, T. and Laine, R. O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival or death. J. Biol. Chem. 277: 4147-4151. 11741912

Andrews, G. K., et al. (2001). The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-1 expression in response to the essential metal zinc in visceral endoderm cells during early development. EMBO J. 20: 1114-1122. 10605938

Apuy, J. L., et al. (2001). Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability. Biochemistry 40(50): 15164-75. 11735399

Auf der Maur, A., et al. (1999). Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol. Chem. 380: 175-185. 10195425

Balamurugan, K., Egli, D., Selvaraj, A., Zhang, B., Georgiev, O., and Schaffner, W. (2004). Metal-responsive transcription factor (MTF-1) and heavy metal stress response in Drosophila and mammalian cells: A functional comparison. Biol. Chem. 385: 597-603. 15318808

Briggs, S. D., et al. (2001). Histone H3 lysine 4 methylation is mediated by SET1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. Genes Dev. 15(24): 3286-3295. PubMed Citation: 11751634

Brugnera, E., et al. (1994). Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1. Nucleic Acids Res, 22: 3167-3173. 8065932

Chen, X., et al. (2004). A novel cysteine cluster in human metal-responsive transcription factor 1 is required for heavy metal-induced transcriptional activation in vivo. J. Biol. Chem. 279(6): 4515-22. 14610091

Cramer, M., et al. (2006). NF-kappaB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells. Biol. Chem. 386(9): 865-72. 16164411

Dalton, T., Fu, K., Palmiter, R. D. and Andrews, G. K. (1996). Transgenic mice that overexpress metallothionein-I resist dietary zinc deficiency. J. Nutr. 126: 825-833. 8613884

Dalton, T. P., Solis, W. A., Nebert, D. W. and Carvan, M. J. (2000). Characterization of the MTF-1 transcription factor from zebrafish and trout cells. Comp Biochem Physiol, B, 126: 325-335. 11007174

Egli, D., Selvaraj, A., Yepiskoposyan, H., Zhang, B., Hafen, E., Georgiev, O. and Schaffner, W. (2003). Knockout of 'metal-responsive transcription factor' MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis. EMBO J. 22: 100-108. 16508004

Egli, D., et al. (2006a). A family knockout of all four Drosophila metallothioneins reveals a central role in copper homeostasis and detoxification. Mol. Cell. Biol. 26(6): 2286-96. 16508004

Egli, D., et al. (2006b). The four members of the Drosophila metallothionein family exhibit distinct yet overlapping roles in heavy metal homeostasis and detoxification. Genes Cells 11(6): 647-58. 16716195

Ercan, S., et al. (2007) X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation. Nat. Genet. 39(3): 403-408. PubMed Citation: 17293863

Giedroc, D. P., Chen, X., Pennella, M. A. and LiWang, A. C. (2001a). Conformational heterogeneity in the C-terminal zinc fingers of human MTF-1: an NMR and zinc-binding study. J. Biol. Chem. 276(45): 42322-32. 11524427

Giedroc, D. P., Chen, X. and Apuy, J. L. (2001b). Metal response element (MRE)-binding transcription factor-1 (MTF-1): Structure, function, and regulation. Antioxid. Redox Signal. 3: 577-596. 11554446

Günes, C., et al. (1998). Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J. 17: 2846-2854. 9582278

Harrison, M. D., Jones, C. E. and Dameron, C. T. (1999) Copper chaperones: function, structure and copper-binding properties. J. Biol. Inorg. Chem. 4: 145-153. 10499084

Heuchel, R., Radtke, F., Georgiev, O., Stark, G., Aguet, M., and Schaffner, W. (1994). The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J. 13: 2870-2875. 8026472

Hsu, D. R. and Meyer, B. J (1994). The dpy-30 gene encodes an essential component of the Caenorhabditis elegans dosage compensation machinery. Genetics 137(4): 999-1018. PubMed Citation: 7982580

Hsu, D. R., Chuang, P. T. and Meyer, B. J. (1995). DPY-30, a nuclear protein essential early in embryogenesis for Caenorhabditis elegans dosage compensation. Development 121(10): 3323-3334. PubMed Citation: 7588066

Kägi, J. H. (1991). Overview of metallothionein. Methods Enzymol. 205: 613-26. 1779825

Langmade, S. J., Ravindra, R., Daniels, P. J., and Andrews, G. K. (2000). The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J. Biol. Chem. 275: 34803-34809. 10952993

Li, Y., Kimura, T., Laity, J. H. and Andrews, G. K. (2006). The zinc-sensing mechanism of mouse MTF-1 involves linker peptides between the zinc fingers. Mol. Cell. Biol. 26(15): 5580-7. 16847313

Lichtlen, P. and Schaffner, W. (2001). Putting its fingers on stressful situations: The heavy metal-regulatory transcription factor MTF-1. Bioessays 23: 1010-1017. 11746217

Marr, M. T., Isogai, Y., Wright, K. J. and Tjian, R. (2006). Coactivator cross-talk specifies transcriptional output. Genes Dev. 20(11): 1458-69. 16751183

Milne, T. A., et al. (2002). MLL targets SET domain methyltransferase activity to Hox gene promoters. Molec. Cell 10(5): 1107-1117. PubMed Citation: 12453418

Murphy, B. J., et al. (1999). Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res. 59: 1315-1322. 10096565

Nagy, P. L., Griesenbeck, J., Kornberg, R. D. and Cleary, M. L. (2002). A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3. Proc Natl Acad Sci 99(1): 90-94. PubMed Citation: 11752412

Nislow, C., Ray, E. and Pillus, L. (1997). SET1, a yeast member of the trithorax family, functions in transcriptional silencing and diverse cellular processes. Mol. Biol. Cell 8(12): 2421-2436. PubMed Citation: 9398665

Palmiter, R. D. (1998). The elusive function of metallothioneins. Proc. Natl. Acad. Sci. 95: 8428-8430. 9671693

Potter, B. M., et al. (2005). The six zinc fingers of metal-responsive element binding transcription factor-1 form stable and quasi-ordered structures with relatively small differences in zinc affinities. J. Biol. Chem. 280(31): 28529-40. 16055450

Puig, S. and Thiele, D.J. 2002. Molecular mechanisms of copper uptake and distribution. Curr. Opin. Chem. Biol. 6: 171-180. 12039001

Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M., Dembic, Z., and Schaffner, W. (1993). Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J. 12: 1355-1362. 8467794

Selvaraj, A., Balamurugan, K., Yepiskoposyan, H., Zhou, H., Egli, D., Georgiev, O., Thiele, D. J. and Schaffner, W. (2005). Metal-responsive transcription factor (MTF-1) handles both extremes, copper load and copper starvation, by activating different genes. Genes Dev. 19(8): 891-6. 15833915

Stuart, G. W., Searle, P. F. and Palmiter, R. D. (1985). Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature 317: 828-831. 4058587

Tamura, Y., et al. (2005). Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1). Oncogene 24(3): 399-406. 15516976

Valls, M., et al. (2000). Drosophila MTN: a metazoan copper-thionein related to fungal forms. FEBS Lett, 467: 189-194. 10675536

Vardanyan, A., et al. (2008). Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila. BMC Dev. Biol. 8: 68. PubMed Citation: 18588663

Wang, Y., et al. (2004). Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. FASEB J. 18: 1071-1079. 15226267

Westin, G. and Schaffner, W. (1988). A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 7: 3763-3770. 3208749

Wimmer, U., Wang, Y., Georgiev, O. and Schaffner, W. (2005). Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione. Nucleic Acids Res. 33(18): 5715-27. 16221973

Yepiskoposyan, H., Egli, D., Fergestad, T., Selvaraj, A., Treiber, C., Multhaup, G., Georgiev, O. and Schaffner, W. (2006). Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc. Nucleic Acids Res. 34(17): 4866-77. 16973896

Zhang, B., Egli, D., Georgiev, O. and Schaffner, W. (2001). The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Mol. Cell. Biol. 21: 4505-4514. 11416130

Zhou, H., Cadigan, K. M. and Thiele, D. J. (2003). A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster. J. Biol. Chem. 278: 48210-48218. 11470482


Biological Overview

date revised: 15 November 2008

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.