Experientia Supplementum Vol. S2Metallothionein II~1987 Birkhauser Verlag Basel

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STRuCruHE AND EXPRESSION OF THE HUMAN METALLOI'HIONElli GENES

L. GedarrDJ., U. Varshney, N. Jahroudi, R. Foster and N.W. Shworak

Department of Biology and the University Biochemistry GroupUniversity of Calgary

Calgary, Alberta, Canada T2N IN4

Abstract: The human metallothioneins are represented by a multigene familyconsisting of about 14 members. A number of MT-like genes have been isolatedfrom a human genomic library and in this report, four MT genes have beencharacterized. Our results show that two of these genes represent the MT-Iand MT-II processed genes. The other two genes (dr-IF and MT-IG) arefunctional members of the MT-I gene family. The amino acid sequence encodedby the MT-I F and MT-IG genes differ from the amino acid sequences of thepublished MT-I proteins at few positions. The S'-flanking region of thesegenes contain metal responsive elements. Our studies show that the MT-IFand MT-IG genes are differentially regulated in two human hepatoma celllines, He 2 and Hep3B2, and a human lymphoblastoid cell line, WI-L2 inresponse to the heavy metals cadmium, zinc and copper, and glucocorticoids.In addition, these genes also show cell-type specific expression.

Introduction

Metdllothioneins (Mrs) are a family of low rrolecular weight ane:> cys­teine rich proteins which bind to heavy rretals such as zinc, cadmium, copperand rrercury (1). They are thought to playa role in either metal detoxifi­cation or ho:reostasis (1). Mrs have been identified throughout a wide spec­trum of living organisms i. e. anirrals, plants and micr=rganisrrs (1). Thepri..rPa.ry structure of the vertebrate Mrs is strongly conc..'erved between species.In particular, the positions of the cysteine residues, which serve to chelateheavy rretal ions via thiolate ccnplexes, are invariant. Mr synthesis is in­duced in the liver and other tissues of anirrals as well as in cell lines whenexposed to heavy rretals or glucocorticoid horrrones (1,2). These proteins arealso develcprentally controlled (3). The inducible nature of these lc.' n::Jlec­ular weight proteins lTBk.es it an ideal system to study the rrolecular rrechan­ism(s) involved in the regulation of eukaryotic gene eA~ression.

'J\...O ITa jor MI' isoforms, denoted. MT-I and MT-TI. have been observedin every vertebrate species examined so far. In the case of humms, thesituation is quite corrplex due to the occurrence of rrany MI'-isoforms.

Subrnlttco \0 the" ccond [nt en tional Meeting on MClallolhionem ano OtherLow olecul r Weight Metal-blrldint; Proteins", Zunch, u"ust 21-2 1" 1985

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There is a single MI'-II p:llypeptide. The MI'-I fraction, hOHever, ShOHS miCl"o­heterogeneity at discrete amino acid p:lsitions and can be separated into atleast 4-5 distinct peaks on high pressure liquid chrc:matography (4). '['he Ml'- J.isoforms av not yet been sequenced; hence, it is not clear wh ther theyrepresent distinct gene products or are the result of p:lst-translational modi­fications.

In an attempt to study the structure, organization, funtion andregulation of the human MI' genes, we have recently screened a hUl1'al1 genol1i.clibrary for clmes containing MI' gene sequences using a heterologous m::>useIT-I cr::NA (5) as a screening probe. We have isolated a number of Mr-lil<e

genes and in this rep:lrt four MI' genes have been characterized by sequing and analysis of their transcripts in three hurnan cell line' and intransfected rrouse cells exposed to heavy !retals and glucocorticoids. ourr suIts ShOH that two of these genes represent the Mr-I and Mr-II processedgenes. The other two genes (MT-IF and Mr-IG) are functional and cxxie fortwo nonallelic rrembers of the r-IT- I gene family. The MI'-IF gene is differ­ent.i lly regulated in resp:lnse to various heavy rretals and glucocorticoids andi s expression appears to be cell-type specific.

Results and Discussion

Gene Family

The human MI's are represented by a multigene family consisting ofabout 14 rrembers ( -8). As sho.m in Fig. 1, hybridization of an EcoRl

'gested gencrnic Southern probed with either TTDuse MJ'-I eDNA or the codingp:lrtion of a hurnan MI'-II processed gene (see Fig. 2) produces several bands.We have attenpted to characterize frBrly of these ban s using hurnan MI'-specificprobes. Based on such studies, the /-IT-I genes oont.aining introns arelocali.zed to the 25.0 kb, 14.5 kb, 10.5 kb, 6.8 kb and 4.5 kb bands (7-9).The 18.0 kb band is represented by a MI'-I processed gene (10, see al Fig.2). The 5.9 kb, 4.8 kb/4.5 kb bands have been identified reviously tocontain the M!'-IIA functional gene and the MI'-II processed gene (6, 10).'!'he other bands, hOHever, may represent additional MT- I processed genes orSCIre MI'-lil<e repetitive sequences because they hybri ize neither to an Ml'-Ispecifi intronic probe (8, 11) nor to /-IT-II specifi_ probe (6).

Our studies and those of others (9, 11) also derronstrate that thMI'- I genes are arranged in tandem. The 25.0 kb and 14.5 kb bands are contig­uo s in the genare and contain the MI'-IG and Mr-IF functi nal genesrespectively (11). Earlier studies have sho.m that the MI'-IA functionalgene is also localized in the 14.5 kb band and that the 14.5 kb band is con­tiguous with the 4.5 kb and 10.5 kb bands containing the MI'-I D gene and theMT-I C and MT-IB functional genes respectively (9). Recently a Mr-Iefunctional gene and another MI'-I pseudogene have been characterized (12).WheL~er these two ge s are closely linked to the MT-I gene cluster describedabove is not kno.m. HOHever, chrarosorra1 localization studies shu.... that IIthe MI'-functiona1 and pseudogenes are on chrarosare 16 (8,13) an theprocessed 9 es are dispersed to several other chrarosares (8, 14). Finally,it appears that Mrs are ceded by five MI'-I and one MT'-II genes.

2-DAYSEXPOSURE

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A B. A 8

~. Study of the Ml'-gene family: Genanic~ fran 2 humms (Aand B) were digested with EcoRI, run on agarose gel, transferred tonitrcx:ellulose paper and hybridised to a nick-translated oodingportion of a gene. The 1lO1ecular weights are fran A rnA digestedwith Hind III.

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MI'- I and MI'-II Processed Genes

During the course of studies esigned to investigate the structureand mechanism of regulation of the hWffiI1 metallothionein rrul igene family, \ole

isolated several hLllTflJ1 MI' genes. '&D of these genes sho.v evidence of RNA-typeprocessing, in that, (i) they lad< introns; (i i) uninterrupted tracts of 17 to21 A residues are ShCMTn to be present, 12-14 bp 3' to the poly (A) signal(AATAAA) of both genes arrl (iii) the presence of directly repeated J::tIlAsequences at the ends of these genes aJ=Pear to define the limits of theinserted CNA fragments. By the aoove criteria these genes are tenred as MI'processed genes (Fig. 2). We have ccnpared the amino acid sequences deducfram the nucleotide sequences of these processed genes with the kno..ln aminoacid sequences of hLllTflJ1 M1'-I and MI'-II polypeptides (15). It is apparent (10)that each processed gene represents the major isofonns of these tx>lypeptides,namely, MT-I and MI'-II.

MI'-II processed gene is highly horologous (95%) in sequence to itscounterpart MT-II gene that has introns which has been ShCMTn to be functional(6). The horology to the functional Ml'-II gene starts exactly at the najorcapsite and ends at its 3' end. A stretch of 17 A residues, ho.vever, arepresent at this tx>int in the MI'-II processed gene. It is very likely that theMI'-II proc ssed gene is derived frcm its functional c.'ounterpart I'fi'-II gene.This processed gene has accurrulated few ITLltations (Fig. 2) callpared to theMI'-II gene. Most of the rrutations in the axling region are s· lent changesexcept for one where a change in amino acid position 10 of the MT-II processedgene seems to be a result of a single base transition form GGT (Gly) in Mr-IIgene to N:J1' (Ser) in the processed gene. on the other hand, the MI'-Iprocessed gene has suffered fran several mutations in tl1e coding protion ofthe gene annng which on creates a prenature termination axlon at amino acidposition 21. Because the sequence of the hWffiI1 MI'-I mRNA or its hmctionalDNA =unt rpart has not been determined, it is not possib to unequivocallyestablish the mRNA start fDint (cap site) of the MT-I processed gene.HOoNever, if one o::xrpares its sequence with that of the MI'-ll processed genesequence and uses the location of the dire t repeats as the limit of th mRNAsequences (confirmed by 51 mapping studies) it appears that Mr-I processedgene is probab y 2-] nucleotides shorter than MT-II processed gene.

Processed genes are related to functional structural genes by virtueof sequence horology and appear to have lost their introns precisely accordingto the rules of RNA splicing (16). A possible xplanation for the generationof th prcx:essed genes involves the rev rse transcription of a processed mRNAto cI:JNA., and then introduct..ionof this CNA copy into the genare via a 5'ov rhanging staggered chrarosaT\3.1 break (17). The details of the rrechanisrnand frequency of these processes are t very well understcxld. Th presenceof precessed genes in ilie genare of various organisms ar;pears to be a camonphenarenon. Ho.vever, the significance of these genes in the organization an:lfunction 0 the genc::me remains unclear. 'The MT-ll prcx:essed gene in hurransShOoNS t\o.Q restriction fragment length {X)lyrrorphisms (RFl.P'sJ of 4.5 kb and 4.8kb when EcoRI digested genanic CNA fran various in . viduals was analysed bySouthern blotting. All the three genotypes are found at a high frequency andthus the I'fi'-II processed gene represen a true polynorphic marker. Familiastudies also indicate that these RFLP's follow the classical mendelianinheritance. Detai.led Southern bio analyses sho.v t this RFLP is due to arestriction site polyrrorphism and is localised at the 5'-flanking region of

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~. Nucleotide sequence of MT-I and MT-II processed genes compared toeach other: The A of ATG, the initiation codon is numbered as 1. Directrepeats (D.R.) at the 5' and 3' ends of the genes as well as the initiationand tennination codons are based. Sequences outside of the direct repeats aresho.om in Sffi3.ll letters whereas capital letters are used to ShON the sequenceof the processed genes. A premature tennination codon present in the MT-Iprocessed gene (61--63) is based with dotted lines. A dash in the codingregion of MT-I processed gene after base 139 represents a deletion mutationcausing a shift in the reading zone.

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the Mr-II processed gene. Sequence analysis of the suspected regi n in the4.8 kb fragrrent ShONS that the sequence G*GA'l1.'C, which is found 371nucleotides <:1ownstream fran the EeaRI site on the 5' -end of the 4.8 kbfragrnant. makes a Hinf I site. A transi ion of *G-A in this sequence ~n the4.5 kb allele has resulted into loss of the Hinf I site and creed an EeaRIsite. Thus. this mutation has given ris to this RFLP. In order to use thisRFLP in the diagnosis of genetically linked diseases, we have perfonnedchromosomal localization studies in collaboration with Dr. F. Ruddl of YaleUniversity. We have lCX'..alized this RFLP. and hence the Mr-II processed gene,on drrarosorre 4 (14). Schmidt et al., (8) als reported its localization onchrarosane 4. The in si.tu hybrid· zation studies have further loc lized thisRFLP in the pll-q2Cregion of chraro ane 4 (14). This RFLP can, th refore, beused as a marker for this reg· on of chromosome 4.

Structure of the hurran Mr-IF and Mr-IG genes

We have also isolated a human genomic clone containing two Mr-Ifunctional genes, Mr-IF and Mr-Ie;. SOUthern blot analyses show that theMT-I F and Mr-IG genes are contained :in the 14.5 kb and 25.0 kb bands of anEeaRI digested genomic DNA repsectively. They are organized in a head-to-headorientation and are located aOO.lt 7.0 kb apart fran each other. The rirra.rystructures of each gene has been C'CI1I)letely determined (11, 18). Like otherMT gen s, /oIT-IF and Mr-IG genes are represented by three exons and tlo.Qintrons. 'l'he location of these :introns i the same as in other MT genes (6,9, 12, 19). HONever, the length of the introns is different than those ofother /oIT--genes. Th first intron 0 the /oIT-lF gene is 582 bp and the se=done is 331 bp ...mile in MT-IG they are 626 bp and 341 bp respectively. Theselengths in hurran Mr-IIA (6) are represented by 301 arrl 203 bp, while inMr-IA ( ) they are 486 bp d 526 bp, respectively. t afPears that thehuman MT-I genes might have evolved with bigger intron lengths. Both of theintrcns are spliced out accordi g to the GT-I\G nlle (16).

'I'he 5' -flanking regions of each gene possess two metal regulatoryelements (MRE) inportant in heavy metal in uction (20-22) and are 70 bp apart.While the MRE in the MT-IF gene ('IR'GCCCGGCCC) is shown to be duplicated,the MT-I G shows 3 base misrratches between the t.1o.Q /omE' s . The sequenceG<XJGGGCGGGTGCAAAG which nay be resp:msible for basal level expression (20) isalso found bet,,;een the MRE's. The 5'-flanking region is also ighly GC rich( 75%) and contains sever l"c..."C" toxes (c;c;c;a;G) probably i.ITp::>rtant in thebinding of transcription factors. '!'he MT-IG has the prq r Tll.TAM OOX andin the MT-IF it is represented by the variant TATCAA sequences. The poJy­adenylation signal sequence, both in the Mr-IF and Mr-IG genes, isrepresented by AATf'AA sequence.

The amino acid sequences of the proteins obtained fran the codingsequences of the /oIT-Ip an /oIT-IG gen s were conpared to the publishedamino acid sequences of the hUl1an MI'-I and Mr-II polypeptides (Fig. 3, 15).The amino acid sequence encoded. by the MT-IF gene differs fran the aminoaci sequences of the Mr-l at four positions. The amino acid positions 11,42, 49 and 61 in the Ml'-IF gen are Val, Ser, Val, an Asp respectively arrlare represented by Gly, Ala, Ile and Ala in the amino aci sequence of theC'fI'-1 protein. On the other hand, the /OIT-IG product sh 5 only two aminoacid differences at positions 11 (Val vs Gly) and 17 (Ser vs Gly). It shouldbe noted that the amino acid sequence derived fran t.l;e MI'-I A gene (9) was

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~. Cooparison of the nucleotide sequences and the amino acid sequences encoded by the hl.lIT'dIlMI'-IF • MI'-IG• MI'-IA (27) and MT-IIA genes to each other and to the amino acid sequencesof the hurran Mr-I and hunan Mr-II polypeptide sequences. The hunan MT-I is heterogeneous and theamino acid positions that sha..; heterogeneity are also sho..m in the ITB.in sequence. Thealternative amino acids are sho..m above the amino acid sequence of the MI'-I polypeptide. Thenucleotide sequences where sho..m by a dash (-) are the same as those of the ~1T-I F gene. Theamino acid sequences where not sho..m are the same as those sho..m by the respective polypeptidesequences. The amino acid sequences encoded by the genes at the MI'-I polypeptide sheAvsrnicroheterogeneity and also the positions where the genes differ fran each other sho..m by thatamino acJ.d. The MT'-I and MI'-II polypeptides are t.1UJT1:bered at an interval of 10 amino acids.

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also found to deviate fran the Ml'- I protei n sequence published prev· ously (15)in the four positi s (amino cid positons 1 , 27, 39 and 40). Thesedifferences are probably due to ambiguity in ele sequence at the protein levelwhich r sults from the difficulties associated with the purification of elevarious MT-I isoforms (4).

In order to classify the MT'-IF and MT-Ie genes, we looked at thnucleotide sequence hamlogies. Both genomic hybridizations and thenucleotide sequences of elese genes shOH close horology to th MT'- I genes(11). Therefore, they were classified as genes belonging to the MT-I family.Moreover, since the MT'-II protein is unique, we had no problem in classifyingthem as MT-I gen s.

Expres ion of the hwren MT'- I F and MT'- IG genes

In order to demonstrate the functionality of the MT'-IF ge1e anJ tostudy its expression without the interference fran other hUlT'al1 genes, we=''1structed a recx::mbinant plasmid containing the Mr-IF and the herpessimplex thymidine kinase (TK) genes and transformed the mouse LMTK- cells.Stable transforrrants were induced with CdC12' ZnC12, CuC12 and dexarretha­sone and total nucleic acids were ass yed for the presence of I-rr-IF mllNA byNorther11 and 51-nuclease analyses using specific probes. TI1e resultsderronstrate that cadmium, zinc and copper but not dexallethasone led to theinduction of the MT'-IF mRNA (data not shOHn). Similar analyses are in theprocess for the MT-I G gene.

The expr ssion of the MT-IF gene was further studied in varioush~1 cell-lines, namely two hepatoma cell lines HepG2 an Hep3B2 (23), and alyrrphobl stoid cell line, WI-1..2 (24). Fran figures 4B and , it is clearthat both cadm.i\.D11, zinc, and copper leod to the induction of the MT'-Ip genein HepG2 cells and is quite similar to elat obta· ned in induced transfornedcells. Massive production of MT'-IF mRNA is obtained in response to all therretals studied. on t.he other hand ele induction of MT'-IF mRNA in Bep3B2 isnot as strong as in HepG2 cells. This differenc in the induction of MT'-Ipgene in the two hepa cell lines nay be attributed to the nature of thesecells (23). Dexarrethasone does not lead to the induction of MT-IF mRNA in00 HeJ=G2 and HepJB2 cells. To our surpris , the MT'-Ip rnRNA is notexpressed in the lyrrphoblastoid cell line (WI-1..2) in response to any of theMr-inducers studied. In order to explain tl1ese results, the expression of theMT-genes in HepG2, Hep3B2 and WI-L2 Was fut-ther investigated. First, totalnucleic acids isolat.ed fran ese cell Ii nes exposed to the various inducerswece hybridized to the coding portion of the /-IT-II processed gene (shOHn tohybridise to all MT'-mRNAs, unpublished) and total /-IT-mRNA was analyzed byNorthern blot teclu1iques. Fig. 4A clearly derronstrates the induction of totaJMT-mRNA in response to cadmium, zinc and capper. Furtherrrore HeJ=G2synth sizes significant levels of rotal MT-mRNA when exposed to dexaJi1:!thasonewhile both WI-1..2 and Rep3B2 cells do not ShOH any induction. These resultsare supported further by our studies on the MT'-polypeptides synthesized inresponse to the various inducers (Fig. 5).

We have also studied the expression of the MT'-IG gene in these cell.­lines using specific probes and o.rr results ShOH th t its expression patternfollows that of the MT-IF gene (18).

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Fig. 4. Expression of the Mr-IF gene: (A) Northern blot analysisof the total nucleic acids extracted form various uninduced i.e.,=ntrol (e) or induced (Cd, Zn, Cu or Dex) cell lines. 10 IIg of thetotal nucleic acids were electrophoresed on a methyl-;rercury hydrox­ide agarose g 1. The RNA was transferred to diazobenzyloxymethylpaper and the filters were hybridised the Mr-II processed genecex:ling regicn (BantiI to PvulI, 10) to detect total Mr- mRNA. Thehuman hepatara cell lines (Hep G2 an Hep 3B2) were induced byeither of the fol owing inducers - 2 1J M CdC12 (Cd), 100 11M Zr1C12(Zn), 100\lM Cl.C12 at' lO\JM dex.arre'thasone (Dex) for 7 hours whilthe lytll)hoblastoid cell line (WI-LZ) was induced for a peric:rl of 12hours. (8) Same as (A) but the filters were hybridised to the 3'­untranslated region of the Mr-IF gene (PvulI - HinfI) to specifi-

370

~ =ntinued-cally detect the t1r-IF mRNA. (C) Sl-nuclease analysis. For chill1alysis 10 g of total nucleic acids were hybridised with 60,000cpm of the 3'-end labelled probe, PvuII - BamHI labelled at thePvuII site, and treated with Sl- nuclease (250 units) and ill1alysedas described else.vhere (10). Hpall fragrnents of pBR322 and HinfIdigest of the Sl-nuclease probe (Hinf) that provided an accuratesize marker for the band of protection is also run along with themarkers. A sample untreated with Sl-nuclease is indicated by (0).Cui represents induction of the Hep 3B2 cells by 100 \J M CuC12 J:

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~. Induction of metallothionein syntheses. Cultures of ahuman lymphoblastoid cell line, \1I-L2 and two human hepatoma celllines, Hep::;2 and Hep3B2, were each expos d to either of the foll~­

ing inducers: 10 lJM De :arnethasone ([)ex), 100 WM CUC12 (Cu), 100 llMZnC12(Zn) and 2 M CdC12(Cd) for 7 hours except WI-L2 where in­duction was for 12 hours. Cells were then labelled for 1 hour with[35S]-cysteine (100 J.lCi/ml, 1100 Ci/rmol) and harvested. Proteinswere car xyrnethylated and analysed on a 20% polyacrylamide gelwithout 8m. A1:xJut 40, 000 cpm per lane was applied. 'J'he uninducedsample. are .indicated by (C).

371

Our studies have sho.om that the MI'-I F and the MI'-~ genes aredifferentially regulated in the Hep32, Hep3B2 and WI-L2 cells in response tothe heavy rretals (Cd, Cu and Zn) and glucocorticoids. In addition to thisdifferential regulation, our studies also ShON that these genes are expressedin a cell-type specific manner. Since these cell-lines have been derived fromdifferent tissues, these studies suggest that these genes shON tissue specificregulation. The finding that the WI-L2 cells do not produce ~fI'-IE' andMr-G mRNAs while xpressing other MI' mRNAs in response to rretals (10) issignificant. our results of the Southern blot analysis of the genanic DNAextracted fran this cell-line ShON that there are no apparent defects in theprirrary t.l-llcture of the genes (unpublished data). We suggest that hyper­rrethylation of the MI'-IF and MI'-IG genes may playa role in the regulationof these genes. In order to address the question of differential and tissuespecific gene expression of the various ~fI'-I genes in detail it is importantthat sequences of other MI'-I genes be detennined and their expression studied.At present we are sequencing t\loO rrore MI'-I genes. OUr future \IoOrk and otherswill be able to answer sane of the possible regulatory rrechanisms.

Ack.n<J..;ledgeIl'eJ1t

This \IoOrk was generously supported by the grants fran MedicalResearch Council (MRC) of Canada, Alberta Heritage Foundation for MedicalResearch (AHFMR) and Provincial Cancer Board (PCHB) to L.G. U.V., N.J. and

.T. were sUp[X)rted by the studentships fran AHFl1R. N.W. Sh<..orak is a Post-doctoral F llON funded by l\HFl'1R. We thank Dr. R.D. Palmiter for his generousgi£t of 01e rrouse MI'-I eDNA clone.

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Dr. Lashitew Gedanu, Department of Biology and the University BiochemistryGroup, University of Calgary, Calgary, Alberta, canada T2N lN4