Regulation of Metallothionein Genes by Heavy Metals Appears to Be

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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1219-1223, February 1994 Biochemistry Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1 RICHARD D. PALMITER Howard Hughes Medical Institute and Department of Biochemistry, SL-15, University of Washington, Seattle, WA 98195 Contributed by Richard Palmiter, November 4, 1993

ABSTRACT A construct, MRE-PGeo, with five metal into cells along with a MRE-driven reporter gene, it resulted response elements fused to a selectable reporter gene was in constitutive expression of the reporter gene in the absence transfected into BHK cells and a stable clone that could be of added metal inducer (16). Thus, MTF-1 is a candidate induced up to 100-fold by zinc, cadmium, bismuth, silver, transcription factor for interaction with MREs, but it is not cobalt, copper, mercury, or nickle was isolated. Some, and clear how it is regulated by metals. The induction of mam- perhaps all, of these metals induce MRE-,Geo by displacing malian MTs by metals is compounded by the observation that zinc. Transfection of these cells with a construct encoding the a wide variety of metals are good inducers, including some transcriptional activator MTF-1 resulted in constitutive ex- metals that are not bound by MT (18). Hence, it is unclear pression of MRE-PGeo, whereas expression of an antisense whether all of these metals stimulate the same traiscription MTF-1 construct in these cells prevented induction by all ofthe factor and, if not, whether different metal-responsive tran- metals. A variant cell line with high conktitutive expression in scription factors recognize different cis-acting elements in the absence of added metals was isolated; normal regulation MT gene promoters. The cloning of MTF-1 (16) provides a was restored by cell fusion. These results suggest that regula- starting point for trying to unravel the regulation of mamma- tion of metallothionein genes by metals is mediated by MTF-1 lian MT genes. interacting with metal response elements and that zinc func- tions to release MTF-1 from an inhibitor. MATERIALS AND METHODS Plasmids. MRE-(3Geo. A BamHI-HindIII fragment con- Metallothionein (MT) genes have been described for a wide taining five MRE-d' elements (6) upstream ofthe basal mouse variety of organisms ranging from yeast to man. These genes MT-I promoter (-42 to +60) was introduced between the Not encode small, cysteine-rich, metal-binding proteins that pro- I and Hind]Il sites of /Geo, a f-galactosidase-neomycin vide protection against metal toxicity and may play a role in phosphotransferase (lacZ-neo) fusion gene (19). CMV- homeostasis ofessential metals (1, 2). There is alto mounting MTF-1. Two pairs of complementary oligonucleotides (25- evidence that they may help protect against reactive oxygen mers with 6 bp of overlap) were synthesized with sequences (3). MT gene expression is ugually inducible. For example, corresponding to the 5' and 3' ends of the published MTF-1 the mammalian MT-I and MT-II genes, which are expressed clone (16). Each oligonucleotide pair was filled in by using in most cells, are induced by a variety of metals, hoimones, DNA polymerase and [a-32P]dNTPs and then combined to and xenobiotics (1, 2, 4, 5). screen a A ZAP mouse brain cDNA library (Stratagene). Five Most MT genes that have been studied are inducible by of nine clones that were plaque-purified hybridized to both metals. Induction of mammalian MT genes by metals is the 5' and 3' probes; the longest one extends 18 bp further 5' mediated by short DNA elements called metal response than the published sequence (16). The Nco I fragment that elements (MREs) that are present in multiple copies in the includes the entire MTF-I open reading frame was inserted promoter region of these genes (5). For example, the mouse between a cytomegalovirus (CMV) promoter/enhancer and a MT-I gene has five functional MREs within the 150 bp simian virus 40 (SV40) polyadenylylation region of a cDNA flanking the transcription start site, but they are not all expression plasmid. Then, a Not I fragment containing the functionally equivalent (6, 7). Genomic footprinting of nuclei CMV-MTF-1-SV40 cassette was introduced into pNUT, a from induced and uninduced cells indicated that a factor is plasmid vector with a dihydrofolate reductase gene driven by bound to the MREs only during induction, suggesting that the SV40 promoter/enhancer (20). CMV-MTF(AZF). The metals allow a positively acting transcription factor to inter- regions encoding the zinc fingers were removed from the act with the MREs (8, 9). Proteins have been detected in construct described above by restriction with ApaLI, which nuclear extracts of mammalian cells that bind to radiolabeled cuts conveniently at the borders of the six fingers, and the MREs, but several different sizes for these proteins have reading frame was restored with an oligonucleotide linker. been reported and the effects of zinc, cadmium, and copper CMV-aMTF-1. When the 2.1-kb Nco I fragment containing on binding activity were variable; thus, the number of dif- the MTF-1 sequence was inserted into the CMV vector, both ferent factors capable of binding MREs is unclear (7, 10-15). orientations were obtained. The antisense orientation was Recently, a clone encoding a MRE-binding protein was moved into pNUT as described above. isolated from a cDNA expression library by using labeled Cells and Transfection. BHK cells (thymidine kinase- MREs as a probe (16). This protein, called MTF-1, has six negative, MT-) were grown in Dulbecco's modified Eagle's zinc fingers of the 2 Cys-2 His type and resembles transcrip- medium (DMEM) (Life Technologies, Gaithersburg, MD) tion factors with this motif (17). It can be isolated in an active plus 10o fetal bovine serum unless otherwise indicated. For form from uninduced cells. When the cDNA for this factor transfection, a calcium phosphate precipitate with 20 ug of was put under control ofa strong promoter and cotransfected plasmid DNA was applied to cells that were about 25%

The publication costs of this article were defrayed in part by page charge Abbreviations: MT, metallothionein; MRE, metal response element; payment. This article must therefore be hereby marked "advertisement" CMV, cytomegalovirus; SV40, simian virus 40; PDC, pyrrolidine in accordance with 18 U.S.C. §1734 solely to indicate this fact. dithiocarbonate. 1219 Downloaded by guest on September 23, 2021 1220 Biochemistry: Palmiter Proc. Natl. Acad. Sci. USA 91 (1994) confluent on a 10-cm plate in 10 ml of medium along with 100 - ,uM chloroquine (21). The medium was changed 4 hr later. 4~~60 60- Cells were split the next day and selection with G418 (800 10t_-. 0 pg/ml) and ZnSO4 (80 pM) or methotrexate (200 pM) was begun. Individual colonies were picked with cloning rings (440 -40 0 about 2 weeks later and then expanded in selection medium for about a month. For some experiments, cells were grown /O in a specially formulated Opti-MEM (Life Technologies) that 0 C(/-'0 was prepared without zinc, Opti-MEMAZn. This medium contains <0.1 zinc compared with 1.0 pLM in normal MM 0 5 10 15 0 0.2 0.4 Q6 0.8 Opti-MEM. DMEM with 10%1 serum contains 3.8 pM zinc. Zinc (/M) Cadmium (pM) Metal concentrations were measured by inductively coupled plasma emission spectroscopy. FIG. 1. Induction of MIRE-,8Geo in cell line 3038-1-1 by zinc and The MRE-,BGeo clone 3038-1-1 was mutagenized six times cadmium. Cells were grown for 3 days in Opti-MEMAZn (e) or in the with ICR-191 (5-10 pg/ml for 2-4 hr) as described by same medium supplemented with 0.5 AM ZnS04 (o) and then the McKendry et aL (22). Then the cells were selected with G418 indicated concentrations of zinc (Left) or cadmium (Right) were at 3.2 mg/ml in the absence of zinc. Clones were picked 2 addedfor an additional 20 hrprior to assay/-galactosidaseof activity weeks later and tested individually for induction of MRE- [(A405 unit per pg of DNA) x 103]. (3Geo by zinc. One of the subclones (3286-8-8) was fused to a hygromycin-resistant clone of BHK cells by plating equal dose-response curves for zinc and cadmium induction of numbers of the two cell types and treating them with 50%1 M[EE-.BGeo in cells grown in Opti-MEMAZn with or without polyethylene glycol for 1 min (23) and then selecting for addition of 0.5 pM ZnS04. Note that these cells were less hybrids with G418 (800 pg/ml), hygromycin (800 pg/ml), and responsive to zinc when grown in synthetic medium contain- ZnSO4 (80 PM). ing 0.5 pM ZnS04 but more responsive to cadmium. The 3-Galactosidase Assay. Cells were trypsinized and plated in maximum extent of induction achieved with several other 24-well dishes 1-2 days before addition of inducers. Stock metals was tested under the same conditions (Fig. 2B) and in solutions of metals (CdSO4, ZnSO4, AgNO3, CuSO4, HgCl2, DMEM plus 10%o fetal bovine serum (Fig. 2A). The concen- bismuth ammonium citrate, CoCl2, NiCl2) or metal chelators tration ofeach metal that gave optimal induction is indicated were diluted about 100-fold into the tissue culture medium. above the bars in Fig. 2; for most metals, higher concentra- About 20 hr later the cells were fixed for 5 min in 0.2% glutaraldehyde and 2% formaldehyde in phosphate-buffered A 160-- saline at 4°C and then rinsed three times in phosphate- 140 3 12 fDMlENP-- C - S buffered saline. One milliliter of 4 mM o-nitrophenyl ,B-ga- lactopyranoside (Sigma) in 25 mM sodium phosphate, pH o120 co 7.7/2mM MgCl2 was added to the fixed cells. The plates were 2? 1001 300 1 0 a 400 incubated at 23°C for 1-2 hr, and the absorbance at 405 nm x was measured. The number of cells was determined by LU 80L suspending unfixed cells in 0.5 ml 0.2% SDS, sonicating 560: briefly, and measuring the amount of DNA by the fluores- a. ' cence of Hoechst 33258 with a TKO-100 fluorometer (Hoe- 20 fer). 150 150 RNA Analysis. Total nucleic acids were harvested by the 0on 1 SDS-proteinase K method (24), followed by phenol/ Cadmium Silver Copper Bismuth Mercury Nickel Cobaltlt.. PDC

chloroform extraction and ethanol precipitation. The samples 160 _- were dissolved and digested briefly with RNase-free DNase. 140KrR\OPTi-MEM Zn " Opti-MEM 0 5 uM Zn The RNA was denatured, electrophoresed through a 1.4% 140 10 agarose gel containing 2.2 M formaldehyde, and then trans- 0.5 ferred to nylon membranes as described (25). The membranes were prehybridized, hybridized with nick-translated lacZ or a-actin probes, and then washed as described (26) except that 100

all washes were at 68°C. x [ii ;.:.!., 0) ~~~~~~100 l.DiiiC RESULTS 60 eli 'i-,i Zinc Induction of MRE-(Geo. Five MRE-d' elements were 0 3200 fused to a basal promoter driving 3Geo, a selectable reporter 2 gene, and transfected into BHK cells, and stable clones were selected in medium containing G418 and ZnSO4. Several .,1t1,1.C.;1 clones were isolated and one of them, 3038-1-1, which was 0 induced about 100-fold by zinc, was chosen for further Cadmium Silver ,;j-ep Bismuth i r5Nickel Cobalt PDC analysis. Southern analysis indicated a single copy of MRE- (3Geo. Half-maximal induction of -galactosidase activity by FIG. 2. Induction of MRE-.8Geo in cell line 3038-1-1 by various zinc was obtained in 6 hr, and maximal activity was reached metals. Cells were grown for 3 days in DMEM plus 10%/ fetal bovine in 20 hr. Upon removal of zinc, (3-galactosidase activity fell serum or (A) Opti-MEMAZn alone or supplemented with 0.5 ,uM ZnSO4 (B); then the cells were treated for 20 hr with five different by halfwithin 10 hr and reached basal levels by 30 hr. For the dosages ofmetals or pyrrolidine dithiocarbonate (PDC) prior to assay experiments described below, cells were split into 24-well of ,-galactosidase activity as shown in Figs. 1 and 4. Maximum dishes so that they would be about 75%M confluent 1-2 days activity relative to that observed with zinc (100%) in the same later, at which time inducers were added. Cells were assayed medium is plotted. The results are the mean of three experiments. for (3-galactosidase activity about 20 hr later. The numbers above the bars are the approximate concentrations Induction of MRE-f3Geo by Various Metals. Fig. 1 shows (uM) of each compound that resulted in optimal induction. Downloaded by guest on September 23, 2021 Biochemistry: Palmiter Proc. Natl. Acad. Sci. USA 91 (1994) 1221 tions were required in the presence of serum. The BHK cells expression of excess MTF-1 leads to constitutive expression used for these studies do not synthesize MT, which increases of MRE-driven reporter genes (16). their sensitivity to some metals. All of the metals tested gave To assess how much MTF-1 mRNA was produced in these some induction in the medium containing serum (Fig. 2A), cells, RNA was isolated for Northern analysis and probed but only zinc, cadmium, bismuth, and silver were effective in with MTF-1 cDNA. The hybridization signal achieved with Opti-MEMAZn. However, when 0.5 pM ZnSO4 was added to 60 ng of RNA from these transfected cells was comparable to the synthetic medium, induction by copper, mercury, and that from 20 pg of control BHK cells. Zinc treatment did not nickel was also observed (Fig. 2B). Hence, all ofthese metals affect the amount of endogenous MTF-I mRNA or that act via transcription factors that recognize MRE-d' and some derived from the CMV-MTF-1 construct. The amount of of them probably induce expression by displacing zinc from MTF-1 mRNA detected in mouse liver, kidney, and spleen other components in the medium. PDC is also an effective was comparable to that in BHK cells; however, there was inducer ofMRE-I3Geo (Fig. 2). Since PDC is a metal chelator, about 50-fold more MTF-1 mRNA in testis (data not shown). it probably acts by increasing the transport of metals into Antisense MTF-1 RNA Blocks Expression of MRE-PGeo. cells. Note that PDC did not induce An antisense construct, CMV-aMTF-1, was made with the MRE-,BGeo in the 2.1-kb Nco I fragment and tested in a manner analogous to synthetic medium unless 0.5 ,uM ZnSO4 was added (Fig. 2B). that described above. Eight methotrexate-resistant clones Constitutive Expression of MRE-fIGeo by MTF-1. The were tested. There was variability among the clones in the coding region of mouse MTF-1 cDNA was cloned next to the extent of inhibition, but in the three best clones induction of CMV promoter/enhancer in an expression vector that also (3-galactosidase activity was only 9-22% ofcontrol cells at all carries a dihydrofolate reductase gene which facilitates se- zinc concentrations tested (Fig. 3). An antisense construct lection of stable clones with high numbers of transgenes (20). was also made with a 690-bp fiagment from the 5' end of the This construct was introduced into the 3038-1-1 cell line and cDNA. Two of the six clones tested inhibited expression five stable clones were selected in the presence of 200 juM about 9-fold (Fig. 3). These results suggest that mouse methotrexate. The induction ofMRE-.BGeo by zinc in each of antisense MTF-1 RNA can effectively block the synthesis of these clones was compared with that in the original 3038-1-1 hamster transcription factor(s) that mediate zinc responsive- clone. All clones responded similarly (Fig. 3). The basal ness. To ascertain whether all the metals that induce MRE- activity in the absence of zinc was higher than that observed ,SGeo act via MTF-1, clone 3256C-5 was tested with each of in the original 3038-1-1 clone when treated with 100 A&M the metals at their optimal concentration in either the rich ZnSO4. MRE-/3Geo expression in all the MTF-1 clones was medium or Opti-MEMAZn. Induction by all metals was induced <1.3-fold by added zinc. These results confirm that inhibited about 10-fold in either medium, suggesting that MTF-1 may be the only factor that binds to MREs (data not shown). MRE-flGeo MREs IacZ neo bGHpA MTF-1 May Be Regulated by an Inhibitory Factor. Because MTF-1 is active when isolated from uninduced cells (16), it Additional Clone Uninduced Maximum may dissociate from an inhibitor during purification. Trans- Construct Activity Zn-induced fection of CMV-MTF-1 into these cells may result in consti- Activity tutive expression by overwhelming the inhibitor. To test this idea, 3038-1-1 cells were mutagenized six times at 2- to 3-week intervals with ICR-191 as described (22), and then none 3038-1-1 1 100 cells that were resistant to G418 at 3.2 mg/ml were selected in the absence of zinc. About 50 clones were picked and _ MTF-1 -SVpA examined for induction of ,-galactosidase by zinc. One CMV-MTF-I CMVp_------clone, 3086-8, had a high basal expression that was stably transmitted to several subclones. Elevated expression in the 3256B-2 172 218 absence of zinc could result from a variety of mechanisms, 3256B-3 200 241 including dominant mutations in transcription factors inter- 3256B-6 156 178 acting with MRE-,8Geo or in the reporter gene itself. To eliminate some of these possibilities, a subclone (3086-8-8) was fused to hygromycin-resistant BHK cells and stable CMCMV-aMTrF-VaMTF- ZCMVp -a---4--anti-MTF-1-4*-. <)SVpA hybrids were selected and then tested for regulation of 2.1 kb (NN) 3256C-5 1 13 MRE-(3Geo. This subclone had a high basal activity in the 3256C-6 3 22 absence of zinc and reverted to normal when cell hybrids 3256C-9 1 9 were prepared (Fig. 4A). The parental clone (3038-1-1) had low basal activity before and after cell fusion (Fig. 4B). Note 0.69 kb (SB) 3269-3 0 13 that the dose-response curve for the variant is shifted to the left relative to the parental cells. Overexpression of MTF-1 CMVp _ MTFROF SVpA produces a similar result (data not shown). Because this CMV-MTF.AZF AZF might reflect a change in zinc transport, the zinc content of the variant cells was compared with that of the parental clone 3283-POP 2 157 by growing them for 3 days in DMEM plus 10% fetal bovine serum with 65Zn (5 ,uM); there was no significant difference FIG. 3. Effect of CMV-driven MTF-1, antisense MTF-I, and in total cellular zinc (data not shown). These results indicate MTF(AZF) on expression of MRE-pGeo. The various constructs that the high basal activity is recessive and suggest that it (see Materials and Methods) were transfected into the 3038-1-1 BHK could be due to the loss of a transcriptional inhibitor. cell line carrying MRE-,3Geo, and stable clones or populations (POP) An MTF-1 construct, CMV-MTF(AZF), that cannot bind of cells resistant to 200 AuM methotrexate were selected. A zinc dose-response experiment was performed as in Fig. 4 and the to DNA was prepared by deleting the zinc-finger domains, maximal P-galactosidase activity achieved relative to the parental with the idea that it might compete with the inhibitor and clone is indicated. bGHpA, bovine growth hormone polyadenylyla- thereby liberate endogenous MTF-1. However, when this tion signal; CMVp, CMV promoter; SVpA, SV40 polyadenylylation construct was transfected into clone 3038-1-1, the induction signal; NN, Nco I-Nco I; SB, Sma I-BamHI. of MRE-f3Geo activity by zinc was normal (Fig. 3). Thus, the Downloaded by guest on September 23, 2021 1222 Biochemistry: Palmiter Proc. Natl. Acad. Sci. USA 91 (1994) lower concentrations of cycloheximide were progressively less effective. This result can be explained by the preferential loss of a transcriptional inhibitor when protein synthesis is blocked.

DISCUSSION The results presented here and the experiments reported by eio 50 Radtke et al. (16) suggest that regulation of mammalian MT genes by heavy metals is fundamentally different from the Ia mechanism worked out for the regulation of the copperthionein gene of Saccharomyces cerevisae. MTF-1 appears to be a constitutively active transcription factor capable of inter- 0 60 1200 60 120 acting with MREs (16). While zinc is undoubtedly essential Zinc (jjM) Zinc for formation of the zinc-finger domain (17), there is no (;jM) evidence that zinc induces folding of the MTF-1 zinc fingers FIG. 4. Properties of a variant cell line with high basal activity. during metal induction, in contrast to the copper-induced The parental 3038-1-1 cell line was mutagenized and a variant folding of the DNA-binding domain of ACE1, the transcrip- (3086-8-8) with high P-galactosidase activity in the absence of zinc tion factor that regulates the copperthionein gene, CUP), in was isolated. Each of these cell lines was fused to hygromycin- yeast (5, 29). resistant BHK cells. (A) ,B Galactosidase activity of the variant cells MTF-1 appears to be the only transcription factor that in response to ZnS04 before (o) and after (o) cell fusion. (B) P-alactosidase activity ofthe parental cells before (o) and after (o) mediates responsiveness to all the metals tested, because cell fusion. Five other hybrid clones ofboth parental and variants had both antisense constructs almost completely inhibit expres- similar induction profiles. Cells were grown in DMEM plus 109o' fetal sion of MRE-(3Geo. Our results suggest a model in which bovine serum; ,B galactosidase activity [(A405 unit per jig ofDNA) x MTF-1 interacts with MREs but it is normally prevented from 103] is indicated. doing so by interaction with an inhibitor that we call MTI, for metallothionein transcription inhibitor (Fig. 6). MTI is the putative inhibitor probably interacts with the zinc-finger metal sensor in this model, but whether the metals interact domain. directly with MTI or indirectly-e.g., by stimulating covalent 'Inducffon of MRE-fiGe mRNA by Cydioheximide. Cyclo- modification of MTI-cannot be deduced from the available heximide, an inhibitor ofprotein synthesis, does not prevent data. Regardless, if this model is correct, then one would induction ofMT-I mRNA by zinc; in fact, there is substantial have to argue that MTI dissociates from MTF-1 during induction of MT-I mRNA by cycloheximide alone (27, 28). biochemical purification, because active MTF-1 can be iso- To ascertain whether this induction might be mediated lated from uninduced cells (16). This model is akin to the through the MREs, the ability of cycloheximide to induce NF-KB and IicB regulatory system (30). The strongest evi- MRE-,BGeo gene expression was tested. Various amounts of dence for this model is the observation that cell variants with cycloheximide were added to the medium for 13 hr. and then high constitutive expression of MRE-,8Geo can be selected RNA was harvested for Northern blot analysis with a lacZ and the regulation reverts to normal when hybrids are formed gene probe. For comparison, some ofthe cells were induced with normal cells. There are other potential explanations for with 100 uM ZnS04 for 13 hr. Fig. S shows that 100 ,uM the high basal activity in the variant cell line, including cycloheximide induced SGeo mRNA about as well as zinc; aberrations in intracellular zinc pools or loss of general factors that influence basal transcription. The 3086-8-8 cell C CHX Z11. line may facilitate cloning genes that can revert the high basal 1 2 3 4 5 6 activity, which would allow a direct test of the model. Transfection with the MTF(AZF) construct resulted in normal regulation of MRE-,fGeo. If this mutant protein was 28S- reasonably stable, these results suggest that neither the N- nor the C-terminal domains of MTF-1 is sufficient to bind to MTI and implicate the zinc fingers in this interaction. The MTF/MTI interaction model has other features that could account for puzzling observations regarding MT gene 1 8S - regulation. For example, inhibition ofprotein synthesis leads to induction of MT gene transcription (27, 28). This is not

MRWs MR-s MRrsIL MT

A% ~~A MT .",Frfmmw

FIG. 5. Effect of cycloheximide (CHX) on expression of BGeo mRNA. BHK cells (clone 3038-1-1) were untreated (lane 1) (control, FIG. 6. Model of MT gene regulation by metals. In the absence C) or treated with 100, 20, 4, or0.8 iM CHX (lanes 2-5, respectively) of zinc, the transcription factor (MTF-1) is complexed with an or 100 ,uM ZnSO4 (lane 6) for 13 hr. RNA was isolated and subjected inhibitor (MTI). MTI dissociates from MTF-1 in the presence ofzinc to Northern analysis. The filter was hybridized with a lacZ probe and allows MTF-1 to interact with MREs in the MT promoter to (Upper) and then stripped and rehybridized with an a-actin probe activate transcription. The MT that is synthesized binds zinc and the (Lower). Positions of 28S and 18S rRNA are shown as size markers. MTF-1/MTI complex reforms. Downloaded by guest on September 23, 2021 Biochemistry: Palmiter Proc. Natl. Acad. Sci. USA 91 (1994) 1223 easily explained if MTF-1 is regulated directly by metals. MTF-I clone and to Phil Soriano for the (3Geo construct. I appreciate However, if MTI has a shorter half-life than MTF-1, then the advice of Helen Blau on cell fusion and the constructive sugges- inhibition of protein synthesis could liberate MTF-1 for tions of my colleagues. This work was supported in part by National transcriptional activation. Another observation that is neatly Institutes of Health Grant HD09172. explained by this model is the constitutive expression of MT genes in certain organs. For example, the highest levels of 1. Kagi, J. H. R. & Kojima, Y. (1987) Experientia Suppl. 52, MT expression in uninduced adult mice are found in testes 25-80. and brain. 2. Suzuki, K. T., Imura, N. & Kimura, M. (1993) Metallothionein Perhaps MTI is not expressed in those organs. III: Biological Roles and Medical Implications (Birkhaeuser, How do different metals cause MTI to dissociate from Basel). MTF-1? A conservative view is that MTI is sensitive only to 3. Tamai, K. T., Gralla, E. B., Ellerby, L. M., Valentine, J. S. & zinc, either because it has binding sites for zinc or because Thiele, D. J. (1993) Proc. Natl. Acad. Sci. USA 90, 8013-8017. zinc activates a system leading to MTI dissociation. Intra- 4. Waalkes, M. P. & Goering, P. L. (1990) Chem. Res. Toxicol. 3, cellular zinc may exist in several pools. It is tightly bound to 281-288. the many zinc metalloproteins; however, it is also weakly 5. Hamer, D. H. (1986) Annu. Rev. Biochem. 55, 913-951. bound to a wide variety of proteins and small molecules. 6. Stuart, G. W., Searle, P. F. & Palmiter, R. D. (1985) Nature Perhaps all the metals capable of inducing MT do so by (London) 317, 828-831. 7. Searle, P. F. (1990) Nucleic Acids Res. 18, 4683-4690. displacing zinc from the weakly bound pool, and this dis- 8. Anderson, R. D., Taplitz, S. J., Wong, S., Bristol, G., Larkin, placed zinc influences the interaction of MTI with MTF-1. B. M. & Herschman, H. R. (1987) Mol. Cell. Biol. 7, 3574- Thus, if MTI responds directly to zinc, it presumably has an 3581. affinity for zinc that is below that of the structural proteins 9. Mueller, P. R., Salser, S. J. & Wold, B. (1988) Genes Dev. 2, but higher than that of the weakly bound zinc. Hence, an 412-427. increase in "free" zinc would lead to liberation of MTF-1. 10. Westin, W. & Schaffner, W. (1988) EMBO J. 7, 3763-3770. The resulting induction of MT would lead to synthesis of 11. Imbert, J., Zafarullah, M., Culotta, V. C., Gedamu, L. & apo-MT that would bind the available zinc and restore the Hamer, D. (1989) Mol. Cell. Biol. 9, 5315-5323. system to equilibrium (Fig. 6). The conceptual simplicity of 12. Anderson, R. D., Taplitz, S. J., Oberbauer, A. M., Calame, K. L. & Herschman, H. R. (1990) Nucleic Acids Res. 18, this model is its most attractive feature. However, variations 6049-6055. on this theme can easily be envisaged. For example, there 13. Sequin, C. & Prevost, J. (1988) Nucleic Acids Res. 16, 10547- may be several different MTIs with different properties, some 10560. metals other than zinc may directly interact with MTI, and 14. Koizumi, S., Suzuki, K. & Otsuka, F. (1992) J. Biol. Chem. MTI may not bind metals directly. 267, 18659-18664. Copper, mercury, nickel, and PDC fail to induce MRE- 15. Czupryn, M., Brown, W. E. & Vallee, B. L. (1992) Proc. Natl. pGeo in Opti-MEMAZn (Fig. 2). However, PDC and copper Acad. Sci. USA 89, 10395-10399. can induce maximal expression, and mercury and nickel can 16. Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M., Dembic, Z. & Schaffner, W. (1993) EMBO J. 12, partially induce MRE-BGeo, when the cells are grown in as 1355-1362. little as 0.5 uM ZnSO4, which by itself results in no expres- 17. Vallee, B. L. (1993) Physiol. Rev. 73, 79-118. sion of MRE-pGeo. Thus, PDC and these metals clearly act 18. Durnam, D. M. & Palmiter, R. D. (1984) Mol. Cell. Biol. 4, by displacing extracellular zinc. The same argument may 484-491. apply intracellularly, where zinc is always present. Cadmium 19. Friedrich, G. & Soriano, P. (1991) Genes Dev. 5, 1513-1523. may also act by displacing zinc. When cells are deprived of 20. Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., zinc they become very sensitive to zinc and relatively insen- Maxwell, I. H. & Brinster, R. D. (1987) Cell 50, 435-443. sitive to cadmium (Fig. 1). The increased sensitivity to zinc 21. Searle, P. F., Stuart, G. W. & Palmiter, R. D. (1985) Mol. Cell. could be due either to increased transport of zinc or to a Biol. 5, 1480-1489. in the 22. McKendry, R., John, J., Flavell, D., Muller, M., Kerr, I. M. & change relative amounts or affinities of MTF-1 and Stark, G. R. (1991) Proc. Natl. Acad. Sci. USA 88, 11455- MTI. The decreased sensitivity to cadmium is predicted if 11459. zinc is the only effective inducer, because during zinc star- 23. Blau, H. M., Chiu, C.-P. & Webster, C. (1983) Cell 32, 1171- vation the low-affinity pool of zinc would be depleted first; 1180. thus, addition of small amounts of cadmium would fill this 24. Durnam, D. M. & Palmiter, R. D. (1983) Anal. Biochem. 131, pool without liberating any zinc. Addition of more cadmium 385-393. would displace zinc from higher-affinity pools. In the case of 25. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular extreme zinc depletion, addition of cadmium leads to cell Cloning: A Laboratory Manual (Cold Spring Harbor Lab. death without induction (data not shown). Press, Plainview, NY), 2nd Ed. 26. Palmiter, R. D., Chen, H. Y. & Brinster, R. L. (1982) Cell 29, In this view, the key to understanding the regulation of 701-710. mammalian MT genes by metals will be the isolation and 27. Mayo, K. E. & Palmiter, R. D. (1981) J. Biol. Chem. 256, characterization ofMTI and determination ofhow it interacts 2621-2624. with MTF-1 and responds to metals. 28. Alam, J. & Smith, A. (1992) J. Biol. Chem. 267, 16379-16384. 29. Furst, P., Hu, S., Hackett, R. & Hamer, D. (1988) Cell 55, I thank Peter Searle for constructing and testing the promoter with 705-717. five MRE-d' elements. I am grateful to Seth Findley for isolating the 30. Baeuerle, P. A. & Baltimore, D. (1988) Science 242, 540-546. Downloaded by guest on September 23, 2021