Dieter Egli

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

ABSTRACT 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. Here we characterize 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 bothDrosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA ofdMTF-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.

Knockout of ‘metal-responsive transcription factor’ MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis

‘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. Here we describe 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 here, 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.

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, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. 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 our 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.

A Family Knockout of All Four Drosophila Metallothioneins Reveals a Central Role in Copper Homeostasis and Detoxification

ABSTRACT 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. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster 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, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midguts “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, we conclude 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. We conclude 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.

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. Here we show, 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, we show that the MtnA promoter is 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.

Copper homeostasis in Drosophila by complex interplay of import, storage and behavioral avoidance

Copper is an essential but potentially toxic trace element. In Drosophila, the metal‐responsive transcription factor (MTF‐1) plays a dual role in copper homeostasis: at limiting copper concentrations, it induces the Ctr1B copper importer gene, whereas at high copper concentrations, it mainly induces the metallothionein genes. Here we find that, despite the downregulation of the Ctr1B gene at high copper concentrations, the protein persists on the plasma membrane of intestinal cells for many hours and thereby fills the intracellular copper stores. Drosophila may risk excessive copper accumulation for the potential benefit of overcoming a period of copper scarcity. Indeed, we find that copper‐enriched flies donate a vital supply to their offspring, allowing the following generation to thrive on low‐copper food. We also describe two additional modes of copper handling: behavioral avoidance of food containing high (⩾0.5 mM) copper levels, as well as the ability of DmATP7, the Drosophila homolog of Wilson/Menkes disease copper exporters, to counteract copper toxicity. Regulated import, storage, export, and avoidance of high‐copper food establish an adequate copper homeostasis under variable environmental conditions.

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

Abstract The zinc finger transcription factor MTF-1 (metalresponsive transcription factor-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 Cu[2+], Zn[2+] and Cd [2+]. 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 stagespecific developmental role. Second, under hypoxicanoxic stress, MTF-1 helps to activate the transcription of the gene placental growth factor (PlGF), an angiogenic protein. Recently we characterized dMTF-1, the Drosophila homolog of mammalian MTF-1. Here we present a series of studies to compare the metal response in mammals and insects, which 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 metalinduced transcript levels are lower. Finally, a clear difference was revealed in the response to mercury, a highly toxic heavy metal: metallothioneintype promoters respond poorly, if at all, to Hg[2+] in mammalian cells but strongly in Drosophila, and this response is completely dependent on dMTF-1.

Physical and functional interaction between the Bloom's syndrome gene product and the largest subunit of chromatin assembly factor 1

ABSTRACT Blooms syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.

Overexpression of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster ameliorates life-span reductions associated with oxidative stress and metal toxicity

Heavy metals are essential components of many biological processes but are toxic at high concentrations. Our results illustrate that when metal homeostasis is compromised by a mutation in the metal-responsive transcription factor (MTF-1), the life-span is shortened. In contrast, MTF-1 overexpression results in resistant flies with prolonged longevity on iron or cadmium-supplemented media but shortened life-span on zinc-supplemented medium. This effect was mediated by the overexpression of MTF-1 in specific tissues, such as the gut, hemocytes and in particular in neurons, indicating that these tissues are particularly sensitive to the perturbance of metal homeostasis. Further, MTF-1 overexpression in a neuron-specific manner protects flies against hyperoxia and prolongs the life-span of Cu/Zn superoxide dismutase-deficient flies, suggesting the presence of a common mechanism for protection against both oxidative stress and metal toxicity. Finally, normal life-span is extended up to 40% upon MTF-1 overexpression in either the peripheral nervous system or motorneurons. These results document the tissue-specific import of heavy metal toxicity and oxidative damage in aging and life-span determination.

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BackgroundMetal-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. 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.ResultsConstitutive 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.ConclusionOur 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.