The Legless Lizard Anguis Fragilis (Slow Worm) Has a Potent Metal-Responsive Transcription Factor 1 (MTF-1)

DOI 10.1515/hsz-2013-0293 Biol. Chem. 2014; 395(4): 425–431

Short Communication

Oleg Georgieva, Viola Günthera, Kurt Steiner, Katharina Schönrath and Walter Schaffner* The legless lizard Anguis fragilis (slow worm) has a potent metal-responsive transcription factor 1 (MTF-1)

Abstract: The metal-responsive transcription factor-1 which lack eyelids, Anguis has eyelids which give its face (MTF-1) is a key regulator of heavy metal homeostasis and a more familiar, seemingly friendly look. Perhaps unaware detoxification. Here we characterize the first MTF-1 from of these differences, Carl von Linné classified Anguis as a a reptile, the slow worm Anguis fragilis. The slow worm, snake; a ‘fragile’ snake, in reference to the typical ability or blind worm, is a legless lizard also known for its long of lizards to shed their tail when attacked by a predator. lifespan of up to several decades. Anguis MTF-1 performs Anguis lives a secluded life in meadows, underbrush and well and matches the strong zinc and cadmium response gardens. A surprisingly long lifespan of several decades has of its human ortholog, clearly surpassing the activity of been documented, but in the wild only a few can expect to rodent MTF-1s. Some amino acid positions critical for live that long; besides losses caused by predators, includ- metal response are the same in humans and slow worm ing domestic cats, slow worms often fall victim to road but not in rodent MTF-1. This points to a divergent evolu- traffic. Unlike other European lizards, which are adept tion of rodent MTF-1, and we speculate that rodents can insect hunters, Anguis mostly feeds on slow prey such as afford a less sophisticated metal handling than humans small slugs, worms and woodlice. and (some) reptiles. Some plants and mushrooms accumulate heavy metals and snails have been reported to have a special Keywords: cadmium toxicity; longevity; metal homeostasis; metallothionein to cope with cadmium load (Palacios metal regulatory transcription factor; nuclear export signal et al., 2011). Along the same vein, woodlice are able to (NES); zinc-induced transcription. accumulate high amounts of heavy metals (Hopkin and Martin, 1982). Therefore we speculated that Anguis fragilis might be particularly well-equipped to cope with aThese auhors contributed equally to this work. heavy metal fluctuations and focused our attention on the *Corresponding author: Walter Schaffner, Institute of Molecular Life Sciences, University of Zürich, CH-8057 Zürich, Switzerland, transcription factor MTF-1 (metal-responsive transcription e-mail: [email protected] factor-1, also referred to as metal-regulatory transcription Oleg Georgiev, Viola Günther, Kurt Steiner and Katharina factor-1, or metal response element-binding transcription Schönrath: Institute of Molecular Life Sciences, University of Zürich, factor-1). CH-8057 Zürich, Switzerland MTF-1 is a main regulator of heavy metal homeostasis in vertebrates (reviewed in Laity and Andrews, 2007; Günther et al., 2012b), and a MTF-1 ortholog has also been The terms slow worm, and especially blind worm, are mis- characterized in the fruit fly Drosophila melanogaster nomers: Anguis fragilis is neither a worm nor is it blind (Zhang et al., 2001; Egli et al., 2003; Balamurugan et al., (Petzold, 1971; Böhme, 1981; Völkl and Alfermann, 2007; 2004). MTF-1 binds via multiple zinc fingers to its cognate Geiser et al., 2013). It is in fact a legless lizard of some DNA motif, termed the metal response element (MRE) 40 cm in length, with a wide distribution centered in the (Stuart et al., 1985; Wang et al., 2004a). MREs share the temperate climate zone of Europe (German: Blindschleiche; core sequence TGCRCNC and are usually found in mul- French: orvet; Italian: orbetto; Spanish: culebra de cristal). tiple copies in the promoter/enhancer region of target The erroneous label ‘blind’ stems from the old Germanic genes. Depending on the type of metal insult, Drosophila plintslîcho/blendschleiche (‘shiny creeper’) and refers to its MTF-1 preferentially binds to specific variants of the MRE skin that appears to glisten as if polished, unlike the scaly, sequence (Sims et al., 2012). The best characterized target more coarse skin of a snake. In contrast to the snakes, genes of MTF-1 encode the metallothioneins, a family of 426 O. Georgiev et al.: Metal responsive transcription factor of slow worm Anguis small, cysteine-rich proteins that bind and sequester a other domains of functional importance that had been variety of heavy metals (Vašák and Meloni, 2011). Mice characterized in human MTF-1 are strongly conserved in with a targeted disruption of the MTF-1 gene die early in the slow worm. Of note, the human nuclear export signal gestation because of liver degeneration (Günes et al., (NES), which is embedded in the acidic activation domain 1998; Wang et al., 2004b) but whether this is caused by a (Saydam et al., 2001; Lindert et al., 2009), is even more defect in metal homeostasis or another function remains similar to the corresponding reptilian sequence of Anguis to be seen. In Drosophila, MTF-1 is dispensable for life (and Anolis) than to the one of the house mouse (Figure 1). under normal laboratory conditions but essential to cope This region is particularly important for metal inducibil- with environmental fluctuations of metal concentrations ity (Lindert et al., 2009). Moreover, the overall length of (Egli et al., 2003). slow worm MTF-1 (738 aa) is similar to Anolis (754 aa) and Here we characterize the first reptilian MTF-1, from the human (753 aa) and thus clearly different from MTF-1 in long-lived lizard Anguis fragilis. We show that its induc- the mouse (675 aa) and another rodent, the South Ameri- ibility upon zinc and cadmium load is as high as that of can capybara Hydrochoerus hydrochaeris (638 aa) (Lindert human MTF-1, and that it clearly outperforms the rodent et al., 2008). The ‘cysteine cluster’ CQCQCAC, which is MTF-1s of mouse and capybara. To obtain Anguis MTF-1, indispensable for MTF-1 activity (Chen et al., 2004; He and a cDNA library was generated from a slow worm tissue Ma, 2009; Günther et al., 2012a) (Figure 1), is also perfectly sample and screened with primers specific for vertebrate conserved in Anguis. MTF-1s. The cDNA coding sequence was assembled and To determine the activity of Anguis MTF-1 we used compared to orthologs of other vertebrates, including the well-characterized Dko7 murine cell line, which human, mouse, capybara and pufferfish fugu (Brugnera carries a targeted disruption of the MTF-1 gene (Heuchel et al., 1994; Auf der Maur et al., 1999; Lindert et al., 2008), et al., 1994). These cells were shown before to work well and of the New World lizard Anolis carolinensis (Alföldi with other transfected vertebrate MTF-1s (Brugnera et al., et al., 2011; Eckalbar et al., 2013). Anguis MTF-1 is a typical 1994; Auf der Maur et al., 1999; Lindert et al., 2008). An vertebrate MTF-1, which not unexpectedly is most closely expression clone of Anguis MTF-1 was tested together related to the other lizard MTF-1. The zinc finger region and with human, mouse, capybara and pufferfish MTF-1 in

Figure 1 Schematic overview of MTF-1 with functionally important domains. Shown here is the domain structure of human MTF-1, which has been studied most extensively. The acidic activation domain, which also includes the nuclear export signal (NES) is known to be essential for activity and metal responsiveness, whereby critical amino acids overlap the NES motif. The NES sequences of human, mouse (Mus musculus), capybara (Hydrochoerus hydrochaeris), slow worm (Anguis fragilis), green anole (Anolis carolinensis), and pufferfish fugu (Takifugu rubripes) are aligned, with divergent amino acid positions indicated in bold red. Note that in this particular region, human MTF-1 is more closely related to reptile than to rodent (mouse, capybara) MTF-1, which is functionally significant [see Figure 4 and Lindert et al. (2009)]. Other domains of functional importance are also indicated (Günther et al., 2012b). For the cloning of Anguis fragilis MTF-1, tissue was retrieved from a roadkill in Weiningen/Zürich, Switzerland, and RNA was isolated using Trizol (Invitrogen) following the supplier’s recommendations. MTF-1 cDNA was assembled from subsegments obtained by the Smart Race cDNA Amplification Kit (Clontech, Saint-Germain-en-Laye, France) and inserted into the expression vector driven by the human cytomegalovirus enhancer-promoter as described (Heuchel et al., 1994). O. Georgiev et al.: Metal responsive transcription factor of slow worm Anguis 427

Figure 2 Vertebrate MTF-1s differ in their transcriptional response to metals. (A) Comparison of the transcription-stimulating activity of human, slow worm (Anguis fragilis), pufferfish, mouse and capybara MTF-1. 0.5 μg of the respective MTF-1 expression plasmids were transfected into MTF-1 knockout cells (Dko7) (Heuchel et al., 1994), together with 10 μg

4 × MREd reporter plasmid and 5 μg reference plasmid per 100 mm dish. If indicated, cells were treated with 100 μm ZnSO4 or 30 μm CdCl2 for 4 h. Reporter and reference transcripts were quantified by the S1 nuclease assay (Weaver and Weissmann, 1979; Westin et al., 1987). The experiment was done in triplicate (bars); gel bands of one representative experiment are shown below. (B) To reveal potential differences in transcriptional activation between human and Anguis MTF-1, transfected Dko7 cells were treated with 50, 100 or 200 μm ZnSO4 or 25, 50 or 100 μm CdCl2 for 4 h (left graph), or 25, 50 or 100 μm ZnSO4 or 10, 25 or 50 μm CdCl2 for 20 h (right graph). (C) Transcriptional activity of human and Anguis MTF-1 in transfected Dko7 cells kept at 37°C or 30°C. White bars, no metal treatment; grey bars, cells exposed to 100 μm

ZnSO4 for 4 h. 428 O. Georgiev et al.: Metal responsive transcription factor of slow worm Anguis cells exposed to zinc or cadmium. As shown in Figure 2A, in the case of mouse MTF-1, less so in capybara, and both human and Anguis MTF-1 responded strongly to both is similar between human and Anguis. We have noted metals, more than the rodent MTF-1s. In another experi- before that transfected mouse MTF-1 is highly expressed ment with human and Anguis MTF-1, exposure to differ- in several cell lines, but at the same time is less metal- ent metal concentrations was extended to 20 h; again the responsive than MTF-1 of human or pufferfish. This was response of both MTF-1s was similar (Figure 2B). Because the case irrespective of whether activity was tested in Anguis is poikilothermic and occasionally exposes itself mouse or human cells. Therefore we asked whether an to sunshine, its body temperature can vary greatly but excessive amount of mouse MTF-1 produced by the trans- is typically lower than that of mammals. Therefore we fected cell, quenches metal inducibility via some auto- wondered if a test at 30°C, rather than the standard 37°C, inhibitory feedback mechanism, such as titration of an would reveal a difference between human and Anguis essential cofactor. This was tested by performing a dilu- MTF-1. This was however not the case: also at the lower tion series of the transfected MTF-1 expression plasmid. temperature both MTF-1s behaved the same (Figure 2C). However, as shown in Figure 3B, the weak metal induc- To verify the production of MTF-1 in the knockout ibility is an intrinsic feature of mouse MTF-1, irrespective host cell, we performed an electrophoretic mobility shift of its expression level. It should nevertheless be pointed assay (Figure 3A). The level of DNA-bound protein is out that the mouse is not defenseless against heavy metal highest for the two rodent MTF-1s, particularly striking load. In vivo, metallothionein gene transcription, which

Figure 3 MTF-1 expression level hardly affects inducibility. (A) Electrophoretic mobility shift assay of human, slow worm (Anguis fragilis), mouse, capybara and pufferfish MTF-1. For binding reactions a radioactively labeled DNA probe containing an MTF-1 binding site was incubated with nuclear extracts (Schreiber et al., 1989) from mouse

MTF-1 knockout cells. These had been transfected with the respective expression plasmids and, if indicated, treated with 100 μm ZnSO4 for 4 h prior to extract preparation. First lane: free probe without nuclear extract added; second lane: nuclear extract of untransfected MTF-1 knockout cells. The electrophoretic binding assays were done according to (Westin and Schaffner, 1988). (B) Human and mouse MTF-1 retain their characteristic inducibility at different MTF-1 concentrations. Dko7 cells were transfected with decreasing amounts of either human or mouse MTF-1 expression plasmid, with other conditions as described for Figure 2, and reporter gene transcripts were quantified by S1 nuclease protection. O. Georgiev et al.: Metal responsive transcription factor of slow worm Anguis 429 strictly depends on MTF-1, is substantially induced by respond to metals (Lindert et al., 2009). Because the dif- cadmium and zinc. What might be missing is a level of ference in metal response between human and mouse functional redundancy that is typical for many biologi- MTF-1 has been mapped to the nuclear export signal/ cal responses. In MTF-1, the zinc fingers are involved in acidic activation domain (Lindert et al., 2009), we com- metal response (Bittel et al., 1998; Zhang et al., 2003) pared the activity of the NES motif of Anguis with that and in humans (and presumably in Anguis) – but not in of its human and mouse counterparts and with selected rodents – the acidic activation domain can independently point mutants. As shown in Figure 4, the NES domains

A B

C D

E F

Figure 4 In contrast to mouse MTF-1, reptile and human MTF-1 harbor a strong nuclear export (NES) function. The NES region of the slow worm Anguis fragilis and corresponding regions of human and mouse (plus two point mutants to make the mouse NES in part more human and/or Anguis-like) were fused to an inert reporter protein and tested for nuclear export function according to (Saydam et al., 2001). NES motifs (see also Figure 1) were: (A) slow worm Anguis fragilis; (B) mouse; (C) human; (D) negative control of reporter protein without NES motif; (E) mouse NES mutation Y→C (SLCLSELGLLST); (F) mouse NES mutation L→M (SLYLSELGLMST). 430 O. Georgiev et al.: Metal responsive transcription factor of slow worm Anguis of both Anguis and human led to the nuclear export of a reptile MTF-1 than to MTF-1 of mouse and capybara. After fused reporter protein. Remarkably, the putative NES of all, the mammalian line diverged from the reptiles more the mouse failed, as did two single point mutations in than 300 million years ago. We also noted that in rodents mouse NES which partly reconstituted a human and/or which tend to be short-lived, MTF-1 is severely truncated Anguis NES sequence. at the C-terminus, indicating that it has evolved faster Do these findings mean that metal inducibility is than in other mammals. We thus speculate that rodents coupled to nuclear export function, such that MTF-1 must can afford a less sophisticated handling of heavy metals shuttle between nucleus and cytoplasm? This scenario than some longer-lived species. is likely an oversimplification, as our previous work has shown that upon inhibition of nuclear export by the Acknowledgments: We thank Drs. Katharina Schmidt and drug LMB, human MTF-1 is confined to the nucleus but George Hausmann for critical reading of the manuscript still retains a large part – though not all – of its metal and for valuable discussions and Dr. Sylvain Ursenbacher inducibility (Lindert et al., 2009). One also has to keep (University of Basel) for information on slow worm biol- in mind that the NES motif overlaps with the acidic acti- ogy. This work was supported by the Swiss National Sci- vation domain, thus a change in one might affect the ence Foundation and the Kanton Zürich. other. In any case we find it striking that at some amino acid positions critical for metal responsiveness, human Received December 10, 2013; accepted January 7, 2014; previously and many other mammalian MTF-1s are more similar to published online January 10, 2014

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