THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 23, Issue of August 15, pp. 11528-11535,1988 Printed in U. S. A.

Structure and Expression of the Human Metallothionein-IG Gene DIFFERENTIAL PROMOTER ACTIVITY OF TWO LINKED METALLOTHIONEIN-I GENES IN RESPONSE TO HEAVY METALS*

(Received for publication, December 23, 1987)

Randy Foster$, Nadia JahroudiS, Umesh Varshneys, and Lashitew Gedamu From the Department of Biological Sciences, University of Calgary, 2500 University Drive N. w., Calgary, Alberta, C a n d a T2N-lN4

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I poly- peptide and has a tripartite structure. The 5”flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3”untranslated region has a variation of the pol- yadenylation signal, AATTAA, and the 3”flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cad- mium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblas- toid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific man- ner. Transient expression studies of the chloramphen- icol acetyltransferase (CAT) gene under the transcrip- tional control of either the hMT-IG or hMT-IF pro- moter in Hep 6 2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT- IGcat in transfected WLL2 cells demonstrates that the hMT-IG promoter is not cell type-specific.

Metallothioneins (MTs)’ are a family of ubiquitous, low molecular weight, cysteine-rich, heavy metal-binding proteins (1, 2). MTs exist as two electrophoretically distinct isoforms, MT-I and MT-11. In man, one MT-I1 and several MT-I isoforms have been identified (3). Although the exact function

* This work was supported in part by a grant from the Medical Research Council (Canada) (to L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequencefs) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession numberfs) 50391 0.

$ Supported by studentships from the Alberta Heritage Foundation for Medical Research.

Alberta Heritage Foundation for Medical Research Postdoctoral Fellow.

The abbreviations used are: MTs, metallothioneins; h, human; CAT, chloramphenicol acetyltransferase; kb, kilobase(s); bp, base pair(s); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; ONPG, o-nitrophenyl-P-D-galactopyranoside; MREs, metal respon- sive elements.

of MTs is not known, they may be involved in heavy metal homeostasis and detoxification, providing protection against hydroxyl free radicals, and, in the acute phase, response to tissue injury and inflammation (4). MT synthesis can be induced by heavy metals and glucocorticoid hormones at the transcriptional level (5, 6) and by a variety of other stressful stimuli (4, 7).

In man, MTs are encoded by a multigene family which consists of at least 12 members (8,9). Of these, four functional MT-I genes, hMT-IA (lo), hMT-IB (ll), hMT-IE (12), and hMT-IF (9, 12), one functional MT-I1 gene, hMT-IIA (8), and several nonfunctional MT genes (8,10,12,13) have been characterized. All of the functional MT genes have been localized to chromosome 16q22 (14, 15). Deletion and muta- tional analysis of the human MT-IIA and mouse MT-I pro- moters have identified a 12-nucleotide motif which mediates heavy metal induction (16-19). Closely related sequences are also present in the transcriptional regulatory regions of other human MT-I genes (9-12).

The expression of these genes has been investigated in a variety of cell lines and tissues (8-12, 20). The hMT-IIA and hMT-IA genes exhibit a ubiquitous expression pattern, al- though the level of hMT-IIA expression exceeds that of the hMT-IA gene (10,ll). The hMT-IB (ll), hMT-IE (12), and hMT-IF (9, 12) genes, however, are expressed in a cell type- specific manner. To gain a better understanding of the molec- ular mechanisms responsible for the transcriptional regula- tion of the human MT-I genes, we have investigated the expression of two tandemly arranged MT-I genes, hMT-IF and hMT-IG. The structure and expression of the hMT-IF gene have been described previously (9). The expression of the hMT-IG gene, like the hMT-IF, hMT-IB, and hMT-IE genes, is cell type-specific. Transfection studies in Hep G2 and Wi-L2 cells using MT-cat fusion genes demonstrate that both the hMT-IG and hMT-IF promoters are functional and responsive to heavy metals and that the hMT-IG promoter is not cell type-specific. Although both promoters exhibit a similar pattern of heavy metal induction, the hMT-IG pro- moter is 5-fold more active than the hMT-IF promoter. The mechanisms which may be responsible for these phenomena will be discussed.

MATERIALS AND METHODS

Cloning of Human MT Genes The isolation of cloned MT-like sequences from a human genomic

library has been described previously (13). DNA from a clone, 14VS, was characterized by restriction endonuclease mapping and Southern blot analyses (9,21). A 4.1-kb Hind111 restriction fragment containing the hMT-IG gene was subcloned into the pUC13 vector and desig- nated phMT-IG.

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Human Metallothionein-IG Structure and Expression 11529

Sequence Analysis

The sequence of a 2.5-kb ApaI-NdeI restriction fragment contain- ing the hMT-IG gene and its flanking regions was determined by the chemical cleavage method of Maxam and Gilbert (22). Restriction fragments with blunt or 3'-overhanging ends were converted to 5'- overhanging ends using T4 DNA polymerase and exonuclease I11 as described previously (9). All the restriction fragments were 5' end- labeled with [T-~'P]ATP and polynucleotide kinase. Labeled frag- ments were electroeluted from agarose gels following secondary diges- tion with the appropriate restriction endonucleases. Complete DNA sequence was confirmed by sequencing both the DNA strands.

Plasmid Constructions MT-IGcat was constructed by blunt end-ligating an end-filled

ApaI-AuaI fragment of hMT-IG (-565 to +65) with an end-filled HindIII digest of pSVOcat (23). MT-IFcat was similarly constructed by ligating a 437-bp XbaI-Bal31 fragment of hMT-IF (-366 to +71) with pSVOcat. The correct orientations were determined by restric- tion mapping and sequence analysis.

Cell Culture, Induction, and DNA Transfection The cell lines derived from a human hepatoblastoma, HepG2, and

hepatocarcinoma, Hep 3B2 (24), were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Flow Laboratories), penicillin (50 units/ml), and streptomycin (5 pglml) at 37 "C in the presence of 5% COZ. The lymphoblastoid-derived cell line, Wi-L2 (25), was grown in suspension in similar medium but was supplemented with 7.5% fetal bovine serum. All cell lines were in- duced by supplementing the growth medium with 2 pM CdC12, 100 pM Cucl2, 100 pM ZnC12, or 10 p M dexamethasone (9).

Plasmid DNA were purified by double CsCl banding. The Hep G2 cells were co-transfected with either MT-IGcat or MT-IFcat con- structs and a reference plasmid, pCHllO (26), which contains a @- galactosidase fusion gene, by calcium phosphate precipitation (27). The cells were plated at a density of 300,000 cells/lOO-mm plate. After 36-40 h of growth the cells were re-fed with medium. Following 1-2 h of incubation, 500 pl of the c%(Po4)2 precipitate containing 15 pg of either MT-IGcat or MT-IFcat, and 10 pg of pCHllO DNA was added to each plate. After 4 h of transfection the medium was removed. The cells were washed with 1 X Hepes-buffered saline (HBS; 0.14 M NaC1, 1.4 mM Na'HPO,, 0.25 M Hepes, pH 7.1), glycerol- shocked (15% glycerol, 1 X HBS) for 1 min, washed with 1 X HBS, and then re-fed with medium. After 24 h of growth the cells were induced with heavy metals for 7 h. These experimental conditions gave optimal CAT activity/pg protein extract assayed.

RNA Preparation and Analysis Transcription Initiation and Poly(A) Addition Sites-For S1 nu-

clease mapping of hMT-IG mRNA, total nucleic acids were prepared from cadmium-induced or -uninduced Hep G2 (9). The transcription initiation site of the hMT-IG gene was delinated by assaying 10 pg of total nucleic acids with 100,000 cpm of an ApaI-AuaI (nucleotides -565 to +65) probe. This probe was derived from a 5' end-labeled 1.1-kb HindIII-AuaI fragment. DNA/RNA hydridizations were car- ried out at 50 "C, digested with 400 units of S1 nuclease (13), and analyzed on a 6% polyacrylamide denaturing gel.

The 3' terminus of the hMT-IG mRNA was defined by hybridizing 10 pg of total nucleic acids with a PuuII-NdeI probe at 45 "C and analyzing the hybrids as described above. This probe was prepared from a 1.1-kb PuuII-PuuII fragment 3' end-labeled with [cY-~'P]~CTP and T4 DNA polymerase (9). The expression of the endogenous hMT- IG and hMT-IF genes in control and heavy metal-induced transfected Hep G2 cells was analyzed by S1 nuclease mapping of the 3' termini of their transcripts. The hMT-IG 3' terminus was mapped using the PuuII-NdeI probe as described above and the 3' terminus of the endogenous hMT-IF gene using a probe encompassing nucleotides 1182-1507 (9). This fragment was excised out of pGEM2-hMT-IF 5'' with EcoRI and HindIII and then 3' end-labeled as described above. All experimental conditions were identical except that hybridizations were done at 40 "C.

Analysis of Transfected Fusion Genes-The transient expression of the MT-IGcat, MT-IFcat, and P-galactosidase fusion genes was ana- lyzed by S1 nuclease mapping. The transcription initiation site of

N. Jahroudi, G. Bietel, R. Foster, and L. Gedamu, manuscript in preparation.

MT-IGcat was delineated by assaying 80 pg of total cytoplasmic RNA (28), prepared from control and metal-induced transfected Hep G2 cells with 50,000 counts of a 5' end-labeled probe. The probe was prepared from a 380-bp PuuII-PuuII fragment of MT-IGcat encom- passing 226 bp of MT-IG 5' sequence (nucleotide -161 to +65) and 150 bp of the CAT structural gene. DNA/RNA hybridizations were carried out at 45 "C. The transcription initiation site of MT-IFcat was similarly determined. Total cytoplasmic RNA (80 pg) was hy- bridized with 50,000 cpm of a 5' end-labeled probe derived from a 520-bp KpnI-PuuII fragment of MT-IFcat. This probe contains 371 bp of hMT-IF 5' sequence and 150 bp of the CAT gene. DNA/RNA hybridizations were carried out at 43 "C. The transcription initiation site of the 0-galactosidase gene was delimited by hybridizing 40 pg of total cytoplasmic RNA with 50,000 cpm of a 5' end-labeled probe. The probe was derived from a 830-bp PuuII fragment of pCHllO which encompasses the 5'-flanking and proximal regions of the 8- galactosidase structural gene. RNA/DNA hybridization was carried out at 48 "C. S1 nuclease digestions were performed as described above and the S1-resistant products analyzed on a sequencing gel.

CAT and Galactosidase Assays Crude cell extracts of transfected Hep G2 cells were prepared as

described by Gorman et al. (23), and the protein concentration was determined using bovine serum albumin as a standard (29). The p- galactosidase activity, used to monitor transfection efficiency of ex- periments, was assayed spectrophotometrically by measuring the conversion of o-nitrophenyl-P-D-galactopyranoside (ONPG) to o-ni- trophenyl at 420 nm. In brief, 40 pl of 4 mg/ml ONPG was added to 30 pg of protein in 200 p1 of 60 mM Na2HP04, 40 mM NaH2P04, 10 mM KC1,l mM MgS04, and 50 mM 8-mercaptoethanol. The reaction was carried out at 37 "C for 1 h and then quenched with 100 p1 of 1 M Na2C03. The @-galactosidase activity was expressed in units where 1 unit = nanomoles of ONPG cleaved/min/mgprotein and nanomoles of ONPG cleaved = A420/0.0045.

To assay for CAT activity, 30 pg of protein extract was adjusted to 120 pl with 0.25 M Tris-HC1, pH 7.8, and then combined with 20 pl of ["C]chloramphenicol (0.133 pCi, 53 mCi/mmol) and 20 pl of 4 mM acetyl-coenzyme A (final concentration, 0.5 mM). The reaction was incubated for 1 h at 37 "C and extracted with 1 ml of ethyl acetate. The ethyl acetate phase was dried out, and the pellet was resuspended in 30 p1 of ethyl acetate. Chloramphenicol and its acetylated deriva- tives were resolved by ascending silica gel thin-layer chromatography for 1.5 h using ch1oroform:methanol (19:l) as a mobile phase and then localized by autoradiography. For quantitation, the spots were cut and the radioactivity determined by liquid scintillation counting. CAT activity was expressed as the percentage of conversion of chlor- amphenicol into its acetylated forms after normalizing to P-galacto- sidase activity.

RESULTS

Sequence Analysis of the MT-IG Gene-The organization of the human genomic DNA clone 14VS is shown in Fig. lA. This clone contains two MT-I genes, hMT-IG and hMT-IF, organized in a head-to-head orientation. The hMT-IG gene is located approximately 7.0 kb downstream of the hMT-IF gene (9). The restriction map of the HindIII fragment con- taining the hMT-IG gene (phMT-IG) is also shown in Fig. lA. Using the sequencing strategy outlined by the underlying arrows, 1.9 kb of DNA sequence encompassing the entire hMT-IG gene was accumulated (Fig. 1B). The amino acid sequence deduced from the DNA sequence demonstrates that this gene codes for a 61-amino acid MT-I polypeptide (1). This polypeptide deviates from the consensus MT-I amino acid sequence at two positions: Val uersm Gly at position 11, and Ser uersm Gly at position 17. The hMT-IG gene has a tripartite structure. The two introns, 626 and 321 bp, respec- tively, occur at the same positions as in all other human MT genes (8-12) and are flanked by the consensus intron splice signal GT . . . AG (30). The lengths of the hMT-IG introns are similar to those of hMT-IF (9, 12) and hMT-IE (12), 585 and 332 bp, and 591 and 348 bp, respectively, but different from those of hMT-IA (lo), hMT-IB ( l l ) , and hMT-IIA (8), 486 and 526 bp, 591 and 480 bp, and 301 and 203 bp, respec-

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11530 Human Metallothionein-IG Structure and Expression

A

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TACCAGGCAA CCTCAGGGAA CCTGCGAAGG CCTACCAGGC TGAGAGAACC CGCACACGCG CCCCACACCC -250

CCTCCCTCCC GAGCCGGTGC GAAAGGGGCC GCCTCCGGTC T G C G C C W E C G C T C A A G CCACCTTGCA -200 -150

Pru I 1

CTTGGCCCAT CTCCTCCGCA CAGCCCAGGC CGGCACCCCG GCCGGTCCGG ACTCAGCCCC CTCGGTGCAA -100

GGGCGCCCCG GGGCCTCTGC GCCCGCCCCC CTCTCCTGAC TATAAAAGCA GCCGCTGGCT GTTGGGCTCC

I LCTCCGCCTT CCACCTCCAC CCACTGCCTC TTCCCTTCTC GCTTGCCAAC TCTAGTCTCG C-TTG

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CAATGGACCC CAACTGCTCC TGTGCCGCTC GTAAGGGACG CCCGCCTTCT CTCCCTTCCl ATGCCAAATT

I50 CCCACACACC ATAGAGAGTC TCCCTGGGTT TGAGGAGGTC GTATTTTSCT ATCACACGTA ACCGCACTCC

200

TTTATTCGTC CAGTCCTTTC CTGTTGGCCA ACCTCCTGAG GGCATTTTCC TCCTCCCTCT TCCTCTATGT

CACAGTTCAG GGTCCTCACC CTCAAGGCTG TCCTGCTCAT CTCAGAGTTG ACGCTCCTCA CCCTCAAGGC 300 350

250

TGTCCTCCCT CACGTCACCT LCTTGGTCAC ACGCCTGCTC CCTCAGCCCC AATTCTCTAA CCTCACTCTG 400

650 AGCTACCGGA TTCCATICGA GACATTCGAT AGCAGGGACA TTGCCTCTTC CAAGTTCAGC ACAGAAAGTC

500 GAAGTCTTCC TAGGCCGTGA T-CCA CTTTCCTTTG GAGTAGAAAT ACCAGCGTCC TTGGTTTTCC

5 I O

? S t 1 600

CACCATCIAT GGIGAGGACA TCGGGCTTCT CTTCCTCGTC TCTGACTGGG AAAC- TGACGGCTGG

CTCTCGCACA CACAAGCGGG AAGTGGACIC TCATTGACCC ACTCCTGTAC CTTCTGCATC TCACTCACCG 700

S r t I b50

L y s C y s T h r S r r C y s L q s L q 55 800 IAATGCACCT CCTGCAAGAA GAGTGAGTGC GGGGCCATCT CCAGGAATCT GGGGCTGTCG CTAAGCTTGG

GACGGAACCC AkGGCTGTCC CTGAGTGCCT GCTTCTGGGG AACCGGCCTT CCTTTGTCCC TGTAGGTTGT 850 900

CACGCCTGTC TAGTCTTCTG CACTTTCCAA CGCTTATGTG ACGTGCCGCA GCTTTCTCAA AGCAACACCC

ATTCCAATGT CCACCAGTTG TCTCCTGACA AAAACCATCC CATCATGAAC TAACGGTCCT CTGGGGCTCC 1050

950

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AGCCATCCAC AC-CT CTTGCGCCAC CGICTTCTAT GATCGAGTCT GCTCTGACCT CTCAATCTCC I100

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I300 TGACCCGTAA AATCTAGGAT TTTTTGTTTT TTGCTICAAT CTTGACCCCT TTCCTACATT CCCTTTTTTC

I 1150 I400 TCTGAAATAT GTGAATAATA AWAAACACT TACACTTGAT TCCCGTTCTG GTTCCTGTTG ;F~???TGGA

ATCAGGGACT GGCGTGGGAG ATTGAACTGG CAGTTCACAC TGGGCTCTGG ACCCAAATGT GAGTCCTAAA 1 4 s o

I500 CAACCTCAGC GCCTTCAGGC ACCCCCGTTA CTTCTCTGAC CTCCTTCCTC TGTAAAAGGC ACCTGCACCG

1550 TGCCGGATGA TATCCGGATC CGGACATACC CG

FIG. 1. Organization and nucleotide sequence of the hMT- IG gene. A, physical structure of the genomic clone 14VS (VS) which contains the hMT-IG and hMT-IF genes. The restriction map of a hMT-IG subclone is illustrated in the lower portion. Open bars represent pUC13 or X Charon 4A sequence and solid bars MT exons. The ouerlying arrows depict the orientation of the linked hMT-IG and hMT-IF genes. The underlying arrows indicate the strategy used to sequence the hMT-IG gene. B, nucleotide sequence of the 5'- flanking, coding, and 3"flanking regions of the hMT-IG gene. The derived amino acid sequence is illustrated above the coding sequence, and the important restriction endonucleases are shown below their respective recognition sequences. The TATAA and ATTAAA se- quences are shown in boldface letters. The transcription initiation and poly(A) addition sites are indicated by arrows. The RNA processing signal, YGTGTTYY, is underscored by circles. Nucleotides upstream of the transcription initiation site are numbered with negative num- bers, whereas those downstream are numbered with positive numbers.

hMT- IG Gene 7 7'

- , ~ ~ ~ ~ r i l l i l i i l i i i i i i i i i i i i i i ) I

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FIG. 2. S1 nuclease mapping of hMT-IG mRNA. The strategy employed to delineate the 5' and 3' limits of hMT-IG transcripts is illustrated in the upper portion. The specific activity of the 5'-specific and 3"specific probes were approximately 3 X 10' and 5 X lo' cpm/ pg, respectively. A, the transcription initiation site was determined by hybridizing an ApaI-AuaI 5' end-labeled probe representing the anti-mRNA strand to total cytoplasmic RNA isolated from cadmium- induced (Cd) or uninduced ( C ) Hep G2 cells. The arrow identifies the 65-bp protected fragment corresponding to the limit of protection upon S1 nuclease digestion. The transcription initiation site repre- sented by an asterisk was identified by matching the size of the protected fragment to a coinciding fragment in the sequencing ladder (lanes C+T and G+A) determined from the AuaI site of the probe. B, the polyadenylation addition site was similarly determined by using a PuuII-NdeI 3' end-labeled probe. The arrow represents the limit of protection and the asterisk the poly(A) addition site as identified by the sequencing ladder determined from the PuuII site of the probe.

tively. The 5' and 3' termini of hMT-IG transcripts were localized by S1 nuclease mapping according to the strategy presented in Fig. 2. The transcription initiation site was localized to an adenine residue, 72 bp upstream of the trans- lation start site (Fig. 2A), and the poly(A) addition site to nucleotide +1363, 7 bp downstream of the polyadenylation signal AATTAA. The 5"flanking region of the hMT-IG gene has a consensus TATAA box 30 bp upstream of the transcrip- tion initiation site. This region is 70% GC-rich and contains four consensus GC motifs, GGGCGG (31), which are located between nucleotides -65 to -60, -70 to -65, -101 to -96, and -298 to -283. This consensus sequence has been shown to be part of the recognition sequence for the transcription factor, SpI (31). We have compared the 5"flanking region of the hMT-IG gene to those of the hMT-IIA and mouse MT-I genes which have had MREs identified by deletion mapping (16-19). hMT-IG possesses four consensus MREs, TGCRCNCGGCCC (la), located between nucleotides -53 to -42, -72 to -83 (in the opposite orientation), -126 to -115, and -144 to -133, and two truncated MREs which contain the first six nucleotides of the consensus sequence located between nucleotides -160 to -154 and -170 to -164. Com-

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Human Metallothionein-IG Structure and Expression 11531

parative sequence analysis of the 3"untranslated and 3'- flanking regions of the hMT-IG gene revealed two RNA processing signals. A variant of the polyadenylation signal, AATTAA (32), is located 125 bp downstream of the transla- tional termination codon. The YGTGTTYY signal (33) lies 26 bp downstream of the polyadenylation addition site (Fig. 1B).

Expression in Human Cells-To address the question of functionality and heavy metal inducibility of the hMT-IG gene, we have examined its endogenous expression in three human cell lines (Hep G2, Hep 3B2, and Wi-L2) which have been shown to express MT mRNA in response to heavy metals (9). S1 nuclease studies using a 5' end-labeled ApaI-AuaI probe were employed to assay hMT-IG transcripts. Fig. 3 demonstrates that hMT-IG is expressed in Hep G2 and Hep 3B2 cells in response to cadmium, copper, and zinc but not to dexamethasone. Since the hMT-IG promoter does not possess a sequence resembling the glucocorticoid responsive element (19) it is not surprising that hMT-IG is not expressed in response to dexamethasone. The pattern of hMT-IG induc- tion, however, differs between the Hep G2 and Hep 3B2 cell lines in response to the various metal ions. In Hep G2 cells, copper and zinc induce maximal accumulation of hMT-IG mRNA while in Hep 3B2 cells cadmium induces the highest level of hMT-IG transcripts followed by zinc and copper, respectively. A longer exposure of the gel was required to detect hMT-IG expression in Hep 3B2 cells in response to copper. Interestingly, the expression of hMT-IG was not detected in Wi-L2 cells. From these results we conclude that hMT-IG, like its linked counterpart hMT-IF, exhibits both cell type-specific and differential expression in response to heavy metals.

Differential Promoter Activity of the MT-IG and MT-IF Genes-Quantitative analysis of hMT-IG and hMT-IF mRNA accumulation in Hep G2 cells indicates that the max- imal inducible expression of hMT-IG is four times that of hMT-IF (34). To gain further knowledge concerning the dif- ferent levels of expression of these two genes, we have fused their promoter regions to the CAT reporter gene (Fig. 4) and analyzer their transient expression in transfected Hep G2 cells. To attain accurate comparisons of CAT activity between experiments, MT-IGcat and MT-IFcat constructs were co- transfected with pCH110, a reference plasmid which encodes the @-galactosidase protein that is under the control of the SV40 early promoter. The transient expression of the MT- ICcat or MT-IFcat and pCHllO fusion genes was analyzed after 7 h of induction with heavy metals. S1 nuclease studies

5

I

FIG. 3. Expression of the hMT-IG gene. S1 nuclease analysis of total nucleic acids extracted from Wi-L2, Hep G2, or Hep 3B2 uninduced (C) cell lines or cell lines induced with cadmium (Cd), copper (Cu), zinc (Zn), or dexamethasone (DX). Wi-L2 cells were induced for 12 h, while the other two cell lines were induced for 7 h. For each analysis, 20 pg of total nucleic acids was hybridized with 20,000 cpm of the ApaI-AuaI 5' end-labeled probe as described under "Materials and Methods." The specific activity of the probe was about 3 X lo8 cpm/pg. The 65-bp S1 nuclease-resistant product is indicated by an arrow. 5' End-labeled HinfI fragments of pBR322 were used as size markers.

Hep G2

C DX C4 Cu Zn

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Hep G2 W i - L 2 Hep 382

C DX C4 Cu Zn C DX C4 Cu Zn C DX Cd Cu Zn

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-300 -200 -100

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MT- IG cat 4 1 " . . . . . . . . 400 -400 -300 -100 -100

FIG. 4. Structure of the MT-IGcat and MT-IFcat fusion genes. The transcription initiation site of each fusion gene is indi- cated by an arrow. Circles represent GC motifs and triangles metal responsive elements.

were employed to investigate the transient expression of the fusion genes at the mRNA transcript level by mapping their transcription initiation sites using 5' end-labeled probes (Fig. 5). Fig. 5A shows that 215 bp of the 380-bp PuuII-PvuII probe is protected by the MT-IGcat transcript upon induction by cadmium, copper, or zinc. A basal level of MT-ICcat mRNA can be detected upon a longer exposure of the gel. Densito- metric scanning of the intensity of S1 protected bands from the MT-IGcat control and metal-induced samples (Fig. 5A) after being normalized to the intensity of their respective 0- galactosidase bands (Fig. 5B) indicates that this construct is differentially induced by the metal ions. Cadmium induces the highest level of MT-IGcat transcript, followed by zinc and copper, respectively. After a 7-day exposure of the gel, Fig. 5 0 shows that MT-IFcat transcripts protect 221 bp of the 520-bp KpnI-PuuII probe upon heavy metal induction. We did not scan the intensity of the S1 protected bands of MT- IFcat transcripts and normalize them to the intensity of their respective &galactosidase bands (Fig. 5E) because of the very high background. These results indicate that the MT-IGcat and MT-IFcat fusion genes are appropriately initiated from the hMT-IG and hMT-IF transcription initiation points and that their expression is inducible by heavy metals. Further- more, comparison of Fig. 5, A and D, taking into account the different exposure times, suggests that MT-IGcat mRNA is more abundant than that of MT-IFcat.

The transcriptional activity of the hMT-IG and hMT-IF promoters was quantitated from the CAT activity generated by the MT-IGcat and MT-IFcat fusion genes. Fig. 6A illus- trates that Hep G2 cells transfected with MT-IGcat exhibit a basal level of CAT activity. In contrast, the basal level of CAT activity generated by MT-IFcat is very low and compar- able to that of the mock-transfected and pSVOcat controls. However, the CAT activity of both MT-IGcat and MT-IFcat can be elevated by metal induction. Of the three metal ions employed, cadmium is the best inducer, followed by zinc and copper, respectively. Comparative analysis of the CAT activ- ity generated by MT-IGcat and MT-IFcat constructs upon

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11532 Human Metallothionein-IG Structure and Expression

A B C

!n Pr C Cd Cu Zn M Pr C Cd Cu Zn ?F M Pr C Cd CI

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MT- IG cat Chimeric Gene @-Galactosidase Fusion Gene 5' 3' 5' 3'

MT- IF cot Chimeric Gene 5' 3'

r\ I:.::.>.::]- . .. . 1 I I 1 . . . .... . . . . . . I

Pvu I[ PVU II: P v u l l Pvu II: Kpn I Pvu II

MT- IG cat mRNA p-Galactosidase mRNA MT-IF cat mRNA

FIG. 5. RNA analysis of transfected Hep 62 cells. S1 nuclease analysis of uninduced (C) or induced (Cd, Cu, or Zn) Hep G2 cells transfected with either MT-IGcat or MT-IFcat and pCH110 as described under "Materials and Methods." The strategy used to probe for the chimeric transcripts is shown below. Arrowheads indicate the S1 resistant fragments. Pr, probes used for each analysis. M , 32P-labeled HinfI digest of pBR322.

normalization to @-galactosidase activity is presented in Fig. 6B and Table I. Both constructs are induced approximately 9-11-fold over the basal level of expression by cadmium, while zinc and copper generate 5-6- and 2-3-fold induction, respec- tively (Table I). Although both constructs exhibit identical -fold induction by heavy metals over their respective basal levels, the level of MT-IGcat expression in all cases is ap- proximately 5-fold that of MT-IFcat.

To compare the transient expression of the MT-IGcat and MT-IFcat constructs to the in vivo situation, we examined the expression of the endogenous hMT-IG and hMT-IF genes in transfected Hep G2 cells. Total cytoplasmic RNA from control and metal-induced Hep G2 cells transfected with MT- IGcat or MT-IFcat and pCHllO were analyzed by S1 nuclease analysis with hMT-IG and hMT-IF 3"specific probes. Fig. 5, C and E, shows that cadmium is the best inducer of the endogenous hMT-IG and hMT-IF genes in Hep 62 transfected with the fusion MT gene constructs. Furthermore, the endog- enous MT genes also respond better to cadmium than the other metals when cells are either mock-transfected or in the presence of pCHllO alone.3 Comparison of Fig. 5 C and F to A and D demonstrates that the endogenous hMT-IG and

hMT-IF genes respond to heavy metals in a qualitative man- ner similar to that of the transfected MT-IGcat and MT- IFcat constructs.

Induction of the hMT-IG Promoter Is Not Cell-type Spe- cific-The observation that hMT-IG is expressed in HepG2 cells but not in Wi-L2 cells (Fig. 3) suggests that hMT-IG is expressed in a cell type-specific manner. In order to investi- gate whether or not the hMT-IG promoter shows cell type specificity, Wi-L2 cells were transfected with the MT-IGcat fusion gene, and CAT activity was measured after metal induction. Our results show that the transfected promoter is inducible and suggest that its expression is probably mediated through the interaction of trans-acting factors (Fig. 7).

DISCUSSION

We have studied the structure and expression of a member of the human MT multigene family, hMT-IG. The sequence and structure of hMT-IG are very similar to that of the other MT-I genes. The promoter has the same potential elements although with positional and copy number variations. hMT- IG, like its linked counterpart hMT-IF, is expressed in a heDatoma-derived cell line but not in a lymphoid-derived cell

R. Foster, unpublished observation. line. Although both hMT-IG and hMT-IF are expressed in a

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Human Metallothionein-IG Structure and Expression 11533 TABLE I

Construct Inducer Average” nor- Induction malizedb

loo#

% conversion‘ -fold

Cadmium 62.3 f 5.8 11.5 Copper 12.9 * 2.6 2.4 Zinc 28.4 k 3.5 5.6

Cadmium 12.4 f 2.9 9.5 Copper 2.6 f 1.2 2.6 Zinc 6.6 f 1.9 5.1

MT-IGcat 5.4 ? 1.4 1

MT-IFcat 1.3 f 1.4 1

a Each measurement is the average of 10 independent CAT assays. ’ Each determination of percent conversion was normalized to 60 units of @-galactosidase activity where: 1 units of @-galactosidase activity = nanomoles of ONPG cleaved/min/mg protein and nano- moles of ONPG cleaved = At~/0.0045.

Percent conversion was determined by dividing the counts/min of all acetylated forms of chloramphenicol by the counts/min of all forms of chloramphenicol.

1

FIG. 6. Comparison of the transient CAT expression of Hep G2 cells transfected with MT-IGcat or MT-IFcat. A, transient expression of MT-IGcat and MT-IFcat fusion genes in response to no inducer ( C ) , cadmium (Cd) , copper (Cu), or zinc (Zn). Chloram- phenicol (CM) and its acetylated forms (A, 1-acetate chlorampheni- col; €3, 3-acetate chloramphenicol) were separated by TLC and de- tected by autoradiography. M, CAT activity of mock-transfected Hep G2 cells. pSV0 , CAT activity of Hep G2 cells transfected with pSVOcat. €3, comparative analysis of MT-IGcat and MT-IFcat CAT activity. CAT activity was expressed as the percentage of conversion of chloramphenicol to its acetylated forms upon normalization to jj” galactosidase activity. The open bars represent no inducer, diagonal striped bars cadmium, vertical striped bars copper, and dotted bars zinc induction. Standard deviation bars calculated from 10 individual trials are shown.

cell type-specific manner, quantitative analysis of the accu- mulation of MT transcripts in Hep G2 cells has indicated that hMT-IG transcripts are more abundant than hMT-IF transcripts (34).

To investigate the transcriptional nature of the differential and cell type-specific expression of the hMT-IG and hMT-IF genes, we have used a transient expression assay to charac- terize their promoter regions. A comparison of the CAT activity generated by the MT-IGcat and MT-IFcat fusion genes in Hep G2 cells has demonstrated that the hMT-IG promoter is significantly stronger than the hMT-IF promoter and that both promoters elicit identical differential induction patterns in response to heavy metals. Similar observations have indicated that the mouse MT-I and MT-I1 genes are coordinately induced by heavy metals (35). In contrast the hMT-IIA and hMT-IA genes have been shown to exhibit a differential expression phenotype in response to heavy metals (10). The expression of MT-IGcat in Wi-L2 cells, a cell line

*!

FIG. 7. Transient CAT expression of Wi-L2 cells trans- fected with MT-IGcat. Transfection was performed using DEAE- dextran as described by Grosschedl and Baltimore (38), and CAT activity was determined as under “Materials and Methods” after inducing cells with metals for 12 h. Other details are as in Fig. 6A.

that does not express hMT-IG, demonstrates that the hMT- IG promoter is responsive to heavy metals and that Wi-L2 cells possess the trans-acting factors necessary to elicit MT- IGcat transcription. This observation indicates that the hMT- IG promoter is not cell type-specific. DNA methylation could be one possible mechanism involved in the control of cell type-specific gene expression. Jahroudi et aL2 have demon- strated that the expression of hMT-IG and hMT-IF is de- tectable in Wi-L2 cells in response to heavy metals after they have been grown in the presence of 5’-azacytidine, an agent which blocks cytosine methylation. Similar observations were reported by Heguy et al. (10) for hMT-IB and Schmidt et al. (12) for hMT-IE. It is possible that the cell type-specific expression of the human MT-I genes may be controlled by DNA methylation of their 5”flanking sequences.

The data presented in this study indicate that the 5’- flanking sequence of the hMT-IG and hMT-IF genes is ac- countable for their similar pattern of metal inducibility and differential promoter strengths. Fig. 8 compares the 5”flank-

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11534 Human Metallothionein-IG Structure and Expression

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ing regions of all the functional human MT-I genes (9-12). We have compared only the proximal 200 bp of 5"flanking sequence, because deletion mapping has shown that only 200 bp of sequence is necessary to elicit differential inducibility and promoter strength of MT-IGcat and MT-IFcat.3 Fig. 8 shows that both the hMT-IG and hMT-IF 5'-flanking se- quences have four consensus metal responsive elements at relatively the same locations: -42 to -53, -62 to -73, -113 to -126, and -131 to -144. The highly conserved spatial organization of these elements may account for the coordinate induction of MT-IGcat and MT-IFcat in Hep G2 cells in response to heavy metals. One difference between the pro- moter elements of hMT-IG and hMT-IF genes is that the former has a true TATAA box and the latter a TATCA box. This difference may in part account for differences in the transcriptional efficiency of the two genes (16). The major differences in the proximal 150 bp of hMT-IG and hMT-IF 5"flanking sequence occur between nucleotides -85 and -115. In this region the MT-IG and MT-IF genes are only 60% homologous as opposed to the 80% homology observed over the rest of the proximal 5'-flanking sequence. The pres- ence of a consensus GC motif in this region, -96 to -101, may contribute to the higher basal level expression of the MT-IGcat construct and account for the stronger promoter strength of the hMT-IG gene. In addition, the two GC motifs and all four MREs present in the proximal 5'-flanking region of the MT-IG gene will reside on the same side of the DNA helix as the TATAA box, assuming 10.5 bp/helical turn. In contrast, one GC box in the 5"flanking proximal region of the MT-IF, -77 to -82, lies on the other side of the helix. Consequently Spl transcription factor binding to the GC motifs in the MT-IG 5'-flanking region could interact with one another and the TATAA box-binding factor with the intervening DNA looping out (37). This putative interaction of trans-acting factors in the MT-IG promoter could account for the higher levels of transcription observed for the MT- IGcat construct. Other investigators have shown that inser- tion of an odd number of multiples of half a helical turn of DNA between the SV40 TATAA box and 21-bp repeats leads to a more drastic decrease in transcription initiation than the insertion of an even number of multiples (36).

I t is interesting to note that the in vivo inducible expression of hMT-IG in Hep G2 cells differs from that in transfected Hep G2 cells. In the in vivo situation, zinc and copper induce hMT-IG to its highest level of expression, while in transfected cells the highest level of induction is observed with cadmium. Although the exact molecular mechanism responsible for this observation is not known, the results of control experiments suggest that this alteration in gene expression may be caused by the transfection process itself. In light of recent studies

- 50

where the role of secondary messengers such as CAMP- and diacylglycerol phosphate-activated protein kinases has been implicated in transcriptional activation, it is reasonable to speculate that calcium may have some role in this pathway. This is supported by the fact that the expression of hMT-IF in response to heavy metals in stable transformants resembles that in Hep G2 cells (9). Perhaps, as a result of the presence of calcium under the conditions of transient expression, cells become more sensitive to the nonphysiological heavy metal inducers such as cadmium and respond by producing large amounts of MT to detoxify its effects.

Although the exact molecular mechanisms involved in the transcriptional regulation of MT genes are not clear, we have presented the first example of differential promoter strength of two linked human MT-I genes. It is possible that the promoters have diverged enough to allow differential recog- nition of their regulatory elements by trans-acting factors. In such a case differential affinity of these trans-acting factors for the regulatory elements may in part mediate the differ- ential promoter strength of these two MT-I genes. It would be interesting to investigate how trans-acting factors interact with either promoter and how these interactions correlate with transcriptional activity.

Acknowledgments-The gift of the pCHllO plasmid from Dr. F. Lee is gratefully acknowledged. We thank Dr. N. W. Shworak for giving the information on Fig. 8 and his help in establishing CAT assays, Dr. C. Sadhu for critical review of the manuscript, and Norma Herrington for typing the manuscript.

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