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).
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).
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date revised: 15 October 2006
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