understanding the mechanisms of zinc-sensing by metal-response element binding transcription...

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ABBArchives of Biochemistry and Biophysics 463 (2007) 201–210

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Understanding the mechanisms of zinc-sensing bymetal-response element binding transcription factor-1 (MTF-1)

John H. Laity a,*, Glen K. Andrews b

a Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USAb Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421, USA

Received 3 January 2007, and in revised form 16 March 2007Available online 4 April 2007

Abstract

The regulation of divalent zinc has been observed in a wide range of organisms. Since this metal is an essential nutrient, but also toxicin excess, zinc homeostasis is crucial for normal cellular functioning. The metal-responsive-element-binding transcription factor-1 (MTF-1) is a key regulator of zinc in higher eukaryotes ranging from insects to mammals. MTF-1 controls the expression of metallothioneins(MTs) and a number of other genes directly involved in the intracellular sequestration and transport of zinc. Although the diverse func-tions of MTF-1 extend well beyond zinc homeostasis to include stress-responses to heavy metal toxicity, oxidative stress, and selectedchemical agents, in this review we focus on the recent advances in understanding the mechanisms whereby MTF-1 regulates MT geneexpression to protect the cell from fluctuations in environmental zinc. Particular emphasis is devoted to recent studies involving theCys2His2 zinc finger DNA-binding domain of MTF-1, which is an important contributor to the zinc-sensing and metal-dependent tran-scriptional activation functions of this protein.� 2007 Elsevier Inc. All rights reserved.

Keywords: MTF-1; Zinc; Metlloregulation; Zinc fingers; Transcription; Zinc-sensing

Zinc is an essential nutrient, but toxic when accumulatedto excess [1–3]. In birds, fishes, and mammals, zinc homeo-stasis and cellular responses to heavy metal toxicity,hypoxia, ionizing radiation, and oxidative stress are regu-lated, in part, by the metal-responsive-element-bindingtranscription factor-1 (MTF-1, Fig. 1a) [4]1 throughmetal-dependent induction of gene expression [3,5–7].Genes regulated by MTF-1 that are metal-inducibleinclude the metallothioneins (MTs) which encode metalion storage proteins [8–10], ZnT1 which encodes a zincefflux transporter protein [11], and glutamate–cysteine

ligase heavy chain (cGCShc) which encodes an oxidative

0003-9861/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2007.03.019

* Corresponding author. Fax: +1 816 235 6584.E-mail address: [email protected] (J.H. Laity).

1 Abbreviations used: MTF-1, metal-responsive-element-binding tran-scription factor-1; MTs, metallothioneins; cGCShc, glutamate–cysteineligase heavy chain; MRE, metal response element; NES, nuclear exclusion;NLS, nuclear localization sequences.

stress-related protein [12]. Many other genes also appearto be regulated by MTF-1, albeit at metal-independent(basal) or limited metal-inducible levels [13–15]. A numberof orthologous genes in Drosophila are likewise regulatedby MTF-1, although zinc-induced transcriptional activa-tion of at least some of the MT genes by MTF-1 is signif-icantly reduced [5,7,16,17]. The importance of MTF-1 isunderscored by its essential role in mouse liver develop-ment [12,18]. While much has been learned about thediverse transcriptional functions carried out by MTF-1since its discovery over 10 years ago [3,4], an understandingof the mechanisms whereby it senses and responds to envi-ronmental stress, and to what extent each stress responsemechanism is coupled, is still emerging.

In this review, we will focus on the molecular mecha-nisms underlying the best understood role of MTF-1 as azinc-dependent transcriptional activator of MT geneexpression [19,20], which is a major regulatory componentin higher eukaryotic zinc homeostasis [10,21]. Particular

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Fig. 1. Domain structure of the mouse MTF-1 protein. (a) The 75 kDa mouse MTF-1 protein has a Cys2His2 zinc finger DNA-binding domain and threetranscriptional activation domains [4]. (b) Primary sequence of the mouse MTF-1 zinc fingers (F) with the metal binding ligands boxed and the consensussecondary structure indicated above the sequence. Numerical positions relative to the start of the canonical helix corresponding to commonDNA-contacting residues [120] are also indicated. The human and mouse sequences are nearly identical, with one conservative R fi K change in thepeptide linker between zinc fingers 4 and 5 underlined [3,4]. (c) Expanded region of MTF-1 which includes the nuclear localization sequence (NLS, redlettering) DNA-binding domain (yellow lettering), acidic domain (blue-grey lettering), and nuclear exclusion sequence (NES, boxed region within theacidic domain). Mouse and human sequences are shown and aligned, but only mouse residue numbers are indicated. Non-conserved residues betweenmouse and human MTF-1 are underlined. (a and b) adapted from [71].

202 J.H. Laity, G.K. Andrews / Archives of Biochemistry and Biophysics 463 (2007) 201–210

emphasis will be placed on recent insights into the molecularmechanisms of MTF-1 zinc-sensing at the level of coordi-nation chemistry within the Cys2His2 zinc finger DNA-binding domain of the protein, and on the role thesespecific zinc-protein interactions play in MTF-1 nuclearlocalization, DNA-binding, recruitment to the mouseMT-I chromatin complex, and subsequent metal-depen-dent activation of mouse MT-I gene expression. Sincecadmium, copper, and oxidative stress-induced MTF-1activation of MT transcription in mammalian and Dro-

sophila cells appear to at least partially involve intracellularzinc redistribution [17,22–25], selected studies involvingthese other heavy metals will also be discussed.

Overview of zinc homeostasis

As the second most abundant essential transition metalion, zinc provides a structural scaffold for many proteinssuch as zinc fingers, zinc clusters, and nuclear hormonereceptors [26–28]. An equally diverse group of functionsare mediated through these metalloproteins, many ofwhich are essential for cellular development and differenti-ation [29]. Zinc also acts as an activator or co-activator inover 300 human proteins from all six enzyme classes [30].

Dietary zinc deficiency has been characterized as a glo-bal health crisis [2,31,32], and numerous studies suggest acorrelation between dietary zinc levels and favorable out-

comes in terms of birth weight and childhood disease inseveral developing countries [33–38]. Zinc deficiency hasbeen linked to abnormal embryonic and fetal development[39], immune dysfunction, neurological problems [40–42],and increased mortality and disease [43,44]. Although notwell understood, zinc toxicity at high intracellular levelscould arise at least partially from unfavorable competitionfor binding sites with other metals in enzymes and metalion transport proteins [10,45,46] and depletion of glutathi-one leading to oxidative stress [12,47–49].

Given its essential but potentially toxic nature, it is notsurprising that the levels of intracellular zinc are tightlycontrolled in prokaryotic and eukaryotic cells[5,11,19,50–62]. Zinc homeostasis is regulated by a complexinterplay of uptake and efflux transporter proteins, coupledwith metal-dependent transcriptional control of selectedtransport and storage proteins [11,48,63–65].

In Escherichia coli, the regulation of intracellular zincappears precise, since the femtomolar affinity reported forthe Zur and ZntR bacterial transcriptional metalloregula-tors suggests that these organisms strive for a level of zincthat exactly matches the number of high affinity metalbinding sites of proteins and other biomolecules withinthe cytoplasm at any given time [50,66]. These observationsled the authors to propose a kinetic mechanism for zinchomeostasis in E. coli possibly involving as yet unidentifiedzinc ion chaperone proteins, rather than one under

J.H. Laity, G.K. Andrews / Archives of Biochemistry and Biophysics 463 (2007) 201–210 203

thermodynamic control that is regulated at the level ofcoordination chemistry [66]. Although kinetic lability ofthe zinc binding sites within a metalloregulatory region ofthe Zap1 transcription factor [56] has also recently beenproposed to be a contributing factor to zinc homeostasisin Saccharomyces cerevisiae [67], zinc affinity measure-ments of metal-sensing domains from the Zap1 andMTF-1 proteins raise the possibility that eukaryotic cellsmay contain significant amounts of labile or accessibleintracellular zinc ions [68–72]. Two separate studiesreported zinc ion affinities in the nanomolar range for thetwo tandem Cys2His2 zinc finger metal sites comprisingthe metalloregulatory transcriptional activation domainof Zap1 [68,69]. MTF-1 also contains a metalloregulatoryDNA-binding domain comprised of six Cys2His2 zinc fin-gers shown in Fig. 1b, which appears to have zinc bindingaffinities in the nanomolar to sub-micromolar range [70–72], and (L. Feng, G.K.A, and J.HL, unpublished results).By contrast, canonical Cys2His2 zinc fingers typically bindzinc with higher affinity (10�9–10�12 M) [73–76]. Consis-tent with the above observations, studies of neuronal cells,where zinc ion concentrations are typically high, reportednanomolar to sub-micromolar accessible intracellular zincion pools [77,78]. Interestingly, mouse MTF-1 can act asa heterologous zinc-responsive transcriptional activator inyeast, which has no chromosomal MTF-1 gene, by activat-ing the expression of an exogenous reporter gene [23]. Thisobservation is consistent with similar zinc-responsive intra-cellular concentrations of accessible zinc ions in yeast andmammalian cells.

Function of MTs and regulation by MTF-1

The functions of MT proteins in higher eukaryotesinclude metal homeostasis, heavy metal detoxification,and protection from oxidative stresses [8,9,79]. Expressionof the mouse MT-I and MT-II genes, for example, is regu-lated in response to these cellular stresses at the transcrip-tional level [4,8,9,48]. Evidence for limited stress responseregulation of MT-IV, which is expressed in squamous epi-thelia and reproductive organs [80], has also been reported.In contrast, mouse MT-III which is expressed in neurons,glia, and male reproductive organs [81–83], does notappear to be inducible. MTs can bind many divalent heavymetal ions including zinc, cadmium, copper, mercury, andcobalt, but MT isolated from mouse tissues is normallyassociated with bound zinc [84,85]. A subset of these metalions (zinc, cadmium, and copper) along with other chemi-cal agents including diethylmaleate, paraquat, nitric oxide,hydrogen peroxide, and cytokines result in significantenhancement of MT gene transcription that is regulatedat the level of transcription [17,86–92]. Induction of MT

genes is mediated by multiple copies of a 12 base pair cis-acting promoter element termed the metal response ele-ment (MRE). The minimal MRE consensus is the highlyconserved (underlined six-base-pair 5 0 region of thesequence: of 5 0-TGCRCnCGGCCC-3 0), while the full-

length MT MRE consensus includes a less-conservedextended GGCCC region on the 3 0 end [93–97].

The MTF-1 transcription factor, which was first clonedfrom mouse [4], has been identified in a wide range ofeukaryotes including humans [3,98], Drosophila melanogas-

ter [5], Takifugu rubripes [57], zebrafish [59,99], trout [59]and chicken (H.M. Jiang and G.K.A, unpublished data).The mouse MTF-1 protein consists of 675 amino acids,and contains a six-Cys2His2 zinc finger DNA-bindingdomain (Fig. 1b) [4], three transcriptional activationdomains (Fig. 1a and c) [4], and putative nuclear exclusion(NES) and localization sequences (NLS) (Fig. 1a and c)[100]. Although mammalian MTF-1 sequences are verysimilar overall (92% sequence identity) [57], the extent ofdomain-specific conservation within MTF-1 orthologs isnot equivalent. The most highly conserved region ofMTF-1 is the zinc finger DNA-binding domain, which isidentical in humans and mouse, with the exception of oneconservative R fi K change in the peptide linker betweenfingers 4 and 5 [3,4] (Fig. 1b). A greater degree of sequencedivergence occurs in the remaining domains of MTF-1.Therefore, many of the observations pertaining to themetalloregulatory function of the highly conserved DNA-binding domain that follow are likely to be generalthroughout all mammalian MTF-1s. Indeed, the zinc-responsive activation of mammalian MT gene expressionmediated by indigenous MRE sites has been demonstratedusing a transfected Drosophila MTF-1 gene [5]. It should benoted that additional as yet uncharacterized zinc or heavymetal-responsive regions within MTF-1 orthologs may bemore functionally variable, reflecting the species-specificdifferences in metal ion homeostasis and stress-responses.

Electrophoretic mobility shift assays (EMSAs) usingwhole cell and nuclear extracts from various higher eukary-otic cell lines, as well as recombinant MTF-1 synthesizedin vitro in a coupled transcription–translation (TnT) sys-tem, demonstrated that MTF-1 binding to the MT-I

MRE is highly sensitive to EDTA and requires low micro-molar concentrations of zinc [19,22,98,101,102]. Further-more, these studies demonstrated that zinc-dependentactivation occurs at 20–37 �C, but not at 4 �C, and DNAbinding is reversibly activated only by zinc. These zinc-responsive DNA binding results are completely consistentwith the reports of nanomolar to micromolar affinitiesfor the Zap1 and MTF-1 zinc finger metal-sensingdomains, given that the equilibrium dissociation constant(Kd) equals the zinc ion concentration when 50% of themetalloregulatory protein binding sites are occupied in asimplified two-state model. However, these in vitro

DNA-binding systems contained numerous other zincbinding proteins in relatively high concentration and anunknown quantity of labile zinc, so a quantitativeassessment of the zinc affinities of the MTF-1 zinc fingersfrom these studies was not possible. Specifically, the poolof accessible zinc ions sensed by MTF-1 and other metallo-regulatory transcription factors originate from a combina-tion of free ions and those in association with other


biomolecules within the cell including the MTs [25]. How-ever, it is also important to note that the non-zinc-regula-tory zinc finger DNA-binding domain from SP1 exhibitedconstitutive in vitro DNA binding in the absence of exoge-nous zinc treatment [103].

Several lines of evidence directly support a parallel zinc-mediated responsive mechanism to cadmium and copperheavy metal loads involving intracellular zinc redistribu-tion and concomitant MT transcriptional activation byMTF-1 in mammalian and insect cells [17,22,24,25].Although these metals have little or no effect on the cog-nate DNA promoter binding activity of MTF-1 in vitro

[22], the stable association of MTF-1 with the chromatincomplex at the mouse MT-I promoter site in vivo is inducedby cadmium and zinc [104], and mutation of the MTF-1metalloregulatory zinc finger DNA-binding domain abol-ishes this induction by both metal ions [104]. Moreover,in vitro cadmium induced transcription of MT-I by MTF-1requires the zinc-loaded form of MT-I (zn7-MT) [25]suggesting that the transcriptional target of MTF-1 itselfprovides a source for zinc induction. However, a veryrecent study using Drosophila demonstrated that MTs arenot required for copper-induced transcriptional activationof MT promotors in vivo [17], which indicates that zinccan be displaced from other lower affinity accessibleintracellular ion pools in this organism.

Molecular mechanisms of zinc-sensing and MTzinc-induction by MTF-1

Experimental evidence described above and elsewheredemonstrates that the zinc-sensing mechanism of MTF-1at least partially involves the six-zinc finger DNA-bindingdomain (hereafter referred to as F1–F6, Fig. 1b). Elucidatingthe molecular details of this mechanism, and more pre-cisely, defining the distinct sensing and other finger-specificfunctions potentially relegated to individual MTF-1 zincfingers within the DNA-binding domain, has been thefocus of a number of research groups since the metallo-regulatory function of MTF-1 was first identified[4,19,70–72,103–108].

Earlier studies of zinc finger peptides derived from theMTF-1 DNA-binding domain reported that 3.5 atoms ofzinc and �12 free thiols were present in different prepara-tions of F1–F6 polypeptides that were subjected to dialysisin the presence of excess Mag-fura-2, supporting the possi-bility of lower affinity sites (Kd P 10�7 M) within a subsetof the MTF-1 zinc fingers [72]. Divalent cobalt-bindingstudies also involving MTF-1 DNA-binding domain zincfinger constructs have suggested subtle context-dependenteffects on metal ion affinities within the six zinc fingers[70]. Specifically, the individual finger cobalt affinitiesincreased �3–20-fold in the full-length F1–F6 protein com-pared to the affinities of the respective isolated fingers [70].However, whether or not these changes in affinities reflectintrinsic and non-cooperative differences of the isolated fin-ger peptides compared to those affinities from the same fin-

ger in the longer six-finger protein could not be resolved inthis study. Overall, an approximately 25-fold cobalt-affin-ity difference was reported for zinc fingers within the intactF1–F6 domain of MTF-1, with the following order of Kd

(tightest to weakest zinc affinity): F4 > F2 @ F5 > F6 @F3 @ F1 [70]. In our recent NMR study of zinc finger pro-teins from the MTF-1 DNA-binding domain, quantitativeanalysis of protein backbone amide NMR 1H-15N-hetero-nuclear single quantum coherence (HSQC) spectral peakintensities measured as a function of added zinc indicatedthat the zinc affinities of all MTF-1 zinc fingers are within�10–50-fold of each other [71,109]. Moreover, the relativezinc ion affinities of the six MTF-1 fingers measured byNMR [71] were in general agreement with those F1–F6cobalt affinities reported earlier [70]. These NMR studiesfurther suggested that zinc-sensing by MTF-1 in eukaryoticcells may involve multiple zinc fingers and occurs over a100-fold or less range of accessible zinc concentration [71].

A fundamentally important question in teasing apart theputative structure/function relationships governing zinc-sensing by a subset of the MTF-1 zinc fingers and the con-comitant transcriptional activation of MT gene expressionby this transcription factor, is whether these processes areregulated at the level of intrinsic zinc binding to individualfinger peptides, or coupled to cooperative finger–fingerinteractions, DNA binding, or a combination of these pos-sibilities. Additional interactions with other factors orMTF-1 domains could also contribute the metalloregula-tory function of the protein. Structural and functionalstudies of DNA binding by MTF-1 zinc fingers havereported conflicting observations relating to which fingersare involved in metal-sensing [71,72,103,105,109–111].Fluorescence polarization DNA-binding studies carriedout by Giedroc and co-workers reported the four N-termi-nal MTF-1 fingers (F1–F4) as the core MRE binding group[111]. Far-UV CD measurements in the same study did notreveal any differences in secondary structure content of fulllength F1–F6 protein in the free and MRE-bound states.Instead, analysis of the CD spectra suggested to theauthors that the MRE undergoes a structural conversionfrom B-form to A-like that is largely mediated by F5 andF6 [111]. From these and other observations, F5 and F6were proposed to be the metalloregulatory fingers thatmediate DNA binding through zinc-induced allostericinteractions with DNA [72,111]. These studies, and laterNMR studies of a truncated F4-F6 MTF-1 zinc finger pro-tein by the same group, provided the experimental basis forthis MTF-1 metalloregulatory model in which F5 and F6bind zinc weakly and F5 does not form a stable bba fold[107,111].

By contrast to this heterogeneous structural model forthe MTF-1 zinc fingers, our recent NMR structural and15N-relaxation data recorded from a F1–F6 protein con-struct demonstrated that each zinc-loaded zinc fingerwithin a polypeptide corresponding to the full-lengthF1–F6 MTF-1 DNA-binding domain adopts a stable bbafold in the presence of stoichiometric zinc, provided that


all cysteine ligands are in a reduced state [71,109]. Simi-larly, analysis of NMR chemical shift data for the MREDNA-bound F1–F6 protein revealed similar folded bbastructures for each MTF-1 zinc finger [71] (and B.M. Pot-ter, L.S. Feng, G.K.A., and J.H.L., unpublished data). Par-allel protein constructs spanning the four N-terminalfingers (F1–F4) and two C-terminal fingers (F5–F6) hadnearly identical stable zinc finger structures free in solution[71]. In both the F1–F6 and F5–F6 proteins, the finger fivecysteines are hyper-sensitive to oxidation at neutral pH[71], which may explain the earlier non-canonical structuralcharacteristics reported for F5 [107]. This oxidation can becontrolled through the addition of TCEP reducing agent,which does not compete for zinc binding [69,112].

Our recent studies also included reduced spectral den-sity [113] and hydrodynamic analysis of NMR 15N relax-ation data [114], which revealed ‘‘quasi-ordered’’anisotropic rotational diffusion properties of the sixF1–F6 zinc fingers [71]. Specifically, the isotropicallyapproximated per finger rotational diffusion values (sc)of 6.9–11.5 ns calculated for MTF-1 zinc-loaded F1–F6and F1–F4 proteins (Fig. 2) were significantly greaterthan the corresponding per finger sc averages of 5–6 nsfor the zinc-loaded DNA-binding domains of the ADR(two fingers) and WT1 (four fingers) transcription factorprotein DNA-binding domains [115,116]. The model thatseems most consistent with the NMR 15N-relaxation datarecorded from the MTF-1 DNA-binding domain is onein which each F1–F6 zinc finger is less domain-flexibleand characterized by significant finger–finger interactions

Fig. 2. MTF-1 zinc fingers are ‘‘quasi-ordered’’. Upper panels, plots of experimback calculated R2/R1 ratios (d) from TENSOR2 [114] as a function of residorientation relative to the axes of diffusion based on the best fit to the 15N-relaxapproximation). Figure adapted from [71].

that produce an elongated ensemble averaged multi-fin-ger conformation. We hypothesize that such interactionscould influence the zinc and DNA-binding properties ofthe MTF-1 zinc-sensing domain [71]. Similar domainordering has been observed previously in a three-zinc fin-ger Xenopus TFIIIA polypeptide [117], which is part of alarger multifunctional DNA and RNA binding domain[73]. Although the MTF-1 15N-relaxation data suggesta high degree of ensemble domain ordering that is spe-cific to this six-zinc finger region, a more general increas-ing trend of sc starting from N- and C-terminal MTF-1zinc fingers that progresses inward to the internal fingers(Fig. 2), suggests for the first time that the intrinsic prop-erty of finger tethering also plays a predictable and moregeneral role in the rotational diffusion properties ofmulti-domain Cys2His2 zinc finger proteins [71]. Specifi-cally, individual domain motions become increasinglymore restricted for internal fingers that are tethered toadjacent single or multiple domains on both ends. Not-withstanding this more general finger position-specificproperty, the above hydrodynamic observations indicatedto us that MTF-1 finger–finger quasi-ordering could bean important determinant in the unusual zinc-sensingfunction of the F1–F6 DNA-binding domain.

Based on these NMR observations and the studiesdescribed below, we now propose a different model forzinc-sensing by MTF-1, in which the F1–F2 zinc fingerpair plays an important role in MTF-1 metalloregulatoryfunction [103,105], while the C-terminal F5 and F6 fin-gers connected by canonical TGEKP linkers appear to

ental R2/R1 ratios (–) that include 5% uncertainty error bars overlaid withue number for each MTF-1 finger, F [71]. Lower panels, possible domaination data. Experimental correlation times (sc) are also indicated (isotropic


be necessary for transcriptional function in the context ofthe chromatin complex [104]. In vivo studies using MRE-driven reporters in dko7 (MTF-1�/�), Drosophila SL2,and yeast cell lines demonstrated that F1 but not F5–F6 of MTF-1 was needed for zinc-inducible activationof DNA binding and subsequent gene expression [103].While in vitro DNA binding of MTF-1 was shown tobe activated in a TnT lysate in the presence of5–15 lM zinc at 37 �C from undetectable levels withoutadded zinc, deletion mutants of MTF-1 missing F1exhibited constitutive, albeit poor, DNA binding in theabsence of exogenous zinc, and were not further acti-vated upon the addition of more zinc [103]. By contrast,no significant difference in zinc-induction or relativeMRE-binding activities were observed in identical assaysof mutant proteins in which F5 or F5–F6 were deletedfrom the MTF-1 zinc finger domain [103]. Moreover, arole for the N-terminal fingers of MTF-1 in zinc-respon-sive transcriptional activity has been reported by Koiz-umi and coworkers [106]. A supporting role for F5 andF6 is suggested from recent studies which demonstratedthat an intact F1–F6 domain is required for MTF-1binding to the MT-I promotor chromatin, and that thisstep is rate-limiting for metalloregulatory function ofthe protein [104].

We recently reported a potential role in MTF-1 zinc-sensing function contributed by the unusual (non-canoni-cal) peptide RGEYT linker connecting the two N-terminal(F1–F2) zinc fingers, and to a lesser extent, the TKEKP lin-ker connecting F2–F3 (Figs. 1b) [105]. A possible zinc sens-ing functional connection between the observed inter-fingerquasi-ordering described previously [71] and the finger lin-ker sequences connecting the four N-terminal MTF-1 fin-gers (F1–F4) was suggested by the high conservation ofthese 4-5 amino acid peptides in mammalian MTF-1 ortho-logs (Fig. 3a) [105]. Moreover, the amino acid compositionof these three N-terminal mammalian MTF-1 finger linkersis highly unusual compared to the canonical TGEKPsequence connecting over 70% of all adjacent Cys2His2 zincfinger motifs [118–120]. Replacing the RGEYT linkerbetween F1 and F2 with TGEKP (L12 MTF-1 mutant)abolished the zinc-sensing function of the protein, resultingin constitutive nuclear translocation, DNA binding, pro-motor recruitment, and transcriptional activation of theMT-I gene (Fig. 3b–e). Swapping the TKEKP linkerbetween F2 and F3 with TGEKP (L23) had more subtlebut similar effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorterTGKT linker between F3 and F4 (L34) rendered MTF-1less sensitive to zinc-dependent MTF-1 functions bothin vivo and in vitro (Fig. 3b–e) [105]. The above observa-tions suggest that the RGEYT finger-linker sequencebetween F1 and F2 and possibly the TKEKP linkerbetween F2 and F3 confer unique properties to theMTF-1 zinc finger domain that allows it to sense changesin available intracellular zinc, and to regulate MT-I geneexpression.

In culture medium containing 10% fetal bovine serumand normal zinc levels, the majority of MTF-1 is locatedin the cytosol, where it must sense changes in zinc availabil-ity [105]. Moreover, incubation of mammalian cells withexcess zinc has also been shown to cause rapid transloca-tion of MTF-1 to the nucleus [100,105,121] and increasedoccupancy of the MREs in the mouse MT-I promoterin vivo [104,122,123]. Although nuclear translocation ofwild-type MTF-1 is rapidly stimulated by the addition oflow micromolar concentrations of exogenous zinc to themedium (Fig. 3b), constitutive nuclear translocation ofthe MTF-1 L12 finger-linker canonical replacement mutantand higher basal nuclear translocation for the MTF-1 L23mutant protein were observed in the absence of exogenouszinc (Fig. 3b) suggesting a link between zinc binding tothese fingers, concomitant ensemble domain structuralordering, and subsequent nuclear translocation [71,105].This model for metal-dependent nuclear localization is con-sistent with the proximity of the NLS and zinc fingerregions of MTF-1 (Fig. 1a and c) [4,100]. More studiesare needed to determine if this process also involves inter-actions with as yet unidentified factors or other regions ofMTF-1.

While available data strongly suggest that the N-termi-nal region of the free, cytoplasmic MTF-1 zinc fingerdomain has a ‘‘de-tuned’’ zinc affinity whereby intracellu-lar zinc concentrations optimal for biological functionscan be responded to, it is ultimately the ternary complexof zinc–MTF-1–DNA in the nucleus that regulates MT-I

gene expression. Given the seemingly greater zinc affinityof the cytosolic MTF-1 L12 and L23 mutants we exam-ined (Fig. 3b), the observed shift in the equilibriumtoward formation of the ternary complex at lower zincconcentrations (i.e., to become a constitutively activetranscription factor) shown in Fig. 3c is expected. How-ever, whether a similar ‘‘de-tuned’’ DNA-bound MTF-1F1–F6 structural arrangement also affects the zinc-proteinaffinity in the ternary complex is still under investigation.Ratiometric pulsed alkylation and mass spectrometrystudies of MTF-1 zinc fingers do suggest that the zincion affinity of some of the fingers may be influenced byDNA binding [108]. Moreover, it is well-established thatcanonical TGEKP zinc finger linkers [118–120] facilitateadjacent finger–finger packing interactions in the DNA-bound state, and contribute to orienting these tetheredfingers along the DNA major grove in such a way as tooptimize specific residue side-chain DNA base interac-tions [124,125]. Interestingly, fine analysis of the finger1-2 RGEYT linker revealed that the two C-terminal resi-dues (Tyr and Thr) are most important for conferringMTF-1 metalloregulatory function [105], which is consis-tent with the known role of these residues in conferringzinc finger packing and orientations in the DNA-boundstate [125]. Molecular level structural and binding studies(zinc and DNA affinities) of the MTF-1 zinc fingers incomplex with the cognate MRE promotor are needed toprovide insights into whether zinc-sensing by the MTF-1

Fig. 3. MTF-1 finger-linker between F1 and F2 is a determinant of metalloregulatory function. (a) Sequences of highly conserved but non-canonical(TGEKP is canonical [118–120]) finger-linker peptides among MTF-1 orthologs from vertebrates and Drospophila. (b) Western blot analysis of metal-induced nuclear translocation of FLAG epitope-tagged wild-type (MTFFL), L12, L23, and L34 finger-linker mutants of MTFFL. Cells were transfectedwith expression vector alone or with the indicated MTF-1FL expression vectors and then incubated with 100 lM ZnSO4 (Zn) or 20 lM CdCl2 (Cd) for 1 h.Cytoplasmic (CE) and nuclear (NE) extracts prepared from the transfected cells were analyzed by Western blotting using an anti-FLAG antibody. (c) Theeffects of mutating zinc finger-linker peptides in MTF-1FL into the canonical linker TGEKP were examined using an electrophoresis mobility shift assay(EMSA). The wild-type, L12, L23, and L34 replacement mutant MTF-1 peptides were synthesized in vitro in a TnT lysate (coupled transcription/translation system) and the effects of the indicated concentrations of zinc on the MREd-binding activity of an aliquot of the lysate were measured. The toppanel shows a larger micromolar zinc range, while the middle panel is expanded on the zinc-responsive range of 0–5 lM. The arrow indicates the specificzinc-MTF-1-MREd complex. The bottom panel shows a control Western blot of the MTF-1FL protein in each TnT lysate reaction. (d) ChromatinImmunoPrecipitation assay (ChIP) analysis of the interaction of MTF-1FL and finger-linker mutants of MTF-1 described in b with the MT-I promoter.Cells were transfected with expression vector alone or with the indicated native MTF-1FL or MTF-1FL finger-linker mutation vectors. Transfected cellswere incubated in medium containing 100 lM ZnSO4 (Zn) or 20 lM CdCl2 (Cd) for 1 h and then chromatin was fixed in vivo, sheared andimmunoprecipitated using anti-Flag agarose beads. The MT-I promoter was amplified by PCR in the precipitated DNA and in the input DNA fromtreated and untreated cells. (e) The effects on the biological activity of MTF-1FL of mutating zinc finger-linker peptides into the canonical linker TGEKPwere examined in transfected KO-MEF cells [105]. The replacement linker mutations described in b were examined. Cells were transfected with vectoralone (lanes 1–3) or with the indicated MTF-1FL expression vectors and then treated with 100 lM ZnSO4 (Zn) or 10 lM CdCl2 (Cd) for 6 h. MT-I mRNAand b-actin mRNAs were detected by Northern blotting. The lower panel is a Western blotting analysis using an anti-FLAG antibody. Figure adaptedfrom [105].


zinc fingers occurs solely at the level of zinc binding to thefree protein, or whether differences in the DNA-boundstructures of the MTF-1 fingers compared to the canoni-

cal zinc finger DNA-binding model [124,125] also plays arole in zinc sensing and metal-inducible transcriptionalactivation functions of MTF-1.


Co-activators and other MTF-1 domains in cellular stress

responses

The above observations do not rule out other regions ofMTF-1, or additional factors as contributors to the zinc-sensing and metal-dependent transcriptional activationmechanisms conferred by MTF-1. Moreover, given thatother regions of MTF-1 are less conserved than the metal-loregulatory DNA-binding domain, some orthologousMTF-1 proteins may be more dependent on other as yetuncharacterized domains for full control of zinc metallore-gulation (most of the studies described in the previous sec-tion used mouse MTF-1). There is also evidence suggestingthe existence of additional regulated steps in MTF-1 heavymetal-induced transcriptional activation of MT. The acidicand proline-rich activation domains depicted schematicallyin Fig. 1a and c have been shown to contribute to metalinducibility, but only in the context of intact MTF-1[126]. A cysteine-rich sequence located in the C-terminalregions found in MTF-1s from pufferfish to humans hasalso been proposed to function as a zinc- and cadmium-responsive domain [127], although no definitive bindingsites within MTF-1 specific for cadmium, copper or otherheavy metals have been identified to support additionalzinc-independent mechanisms for heavy metal inductionof MT. Finally, recent DNA selection and amplificationexperiments by Schaffner and co-workers raise the possibil-ity that DNA base variability within the less conserved 3 0

region of the MRE may play a role in differentiatingmetal-induced and basal MTF-1 transcriptional targets[97].

MTF-1 has a variety of stress response functions inaddition to zinc homeostasis and heavy metal detoxifica-tion, many of which may involve distinct mechanismsand signaling cascades. Accordingly, MTF-1 integrates adiverse set of environmental signals, and modulates eitherdirectly or indirectly a wide array of genes [14,128,129].Indeed, the potential for diverse functions of MTF-1 ishighlighted by the enhanced sensitivity of MTF-1-knock-out Drosophila flies to copper starvation [17], and theMTF-1-dependent repression of basal expression of themouse Slc39a10 gene [15], which encodes for the putativeZIP zinc influx transporter protein. While the details ofthese and other MTF-1 mechanisms are unknown, evi-dence of additional co-factor involvement is supported byrecent observations that MTF-1 directly interacts with orcooperates with a multitude of factors, including NF jB[130], HIF-1a [131], USF [132], SP1 [133], HSF-1[134,135], and ribosomal protein S35 [136]. It has been sug-gested that MTF-1 is a target for regulation by phosphor-ylation [137], but gene- and cell-type-specific effects ofsignal transduction cascades on metal-regulated gene tran-scription appear to be independent of changes in the phos-phorylation of MTF-1 [138].

Recent progress has advanced our understanding of thediversity of MTF-1 functions, and considerably clarifiedthe mechanism whereby MTF-1 senses and responds to

changes in intracellular zinc loads (and possibly otherheavy metal loads and oxidative stresses through an indi-rect redistribution mechanism). However, future studiesof the MTF-1 protein complex are required to furtherdelineate the mechanisms and co-factor interactions under-lying its modulation of numerous target gene expressionprofiles in response to a broad array of environmentalstresses.

Acknowledgments

We acknowledge support from the National Institutes ofHealth (R01 ES05704 to G.K.A.) and the University ofMissouri Research Board (J.H.L.) for funding of selectedstudies described in this review. We thank L.S. Feng forassistance in manuscript preparation.

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