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-1^140-1R) 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 (Ctr1B^3-4) 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-1^140-1R) 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 UAS_GAL. 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 Ctr1B^11-19, 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).

GENE STRUCTURE

cDNA clone length - 3343

Bases in 5' UTR - 180

Exons - 3

Bases in 3' UTR - 787

PROTEIN STRUCTURE

Amino Acids - 791

Structural Domains

Searching through Drosophila databases, open reading frames related to MTF-1 were found in EST sequences derived from adult fly heads and from Schneider cells. However, the MTF-1 candidate sequence reconstructed from EST clones lacked the first zinc finger and also lacked any reasonable in-frame start codon. The complete N-terminal region could only be deduced after isolation and analysis of clones from an embryonic cDNA library. The intron-exon structure of dMTF-1 reveals three introns; the last one is at a position identical to that for the one in vertebrates, while the other two intron positions are unique to Drosophila. To determine more accurately the transcription start site, an extension reaction (RACE) was performed and also the 5' end of transcripts was determined independently by transcript mapping with nuclease S1. These data are consistent and indicate a transcription start downstream of a putative TATA box and a 3.4-kb mRNA for dMTF-1. dMTF-1 cDNA encodes a protein of 791 amino acids, which is slightly larger than vertebrate MTF-1. A comparison of the protein sequences between the three vertebrate species and Drosophila reveals a particularly striking similarity in the region of all six zinc fingers. The similarity is 78% in the zinc finger region and 27% outside it, i.e., 39% in the total protein. In order to verify the data obtained by sequencing and transcript mapping, a Northern blot was performed with samples of Drosophila poly(A) RNA; this indicated a steady increase of dMTF-1 mRNA 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).

date revised: 15 October 2006

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