Metallothioneins (MTs) ()are low molecular mass (6-7 kDa), conserved proteins that coordinate metals, such as cadmium, copper, and zinc, by thiol bonds with their numerous cysteine residues(1) . Although the precise function(s) of MTs has not been defined, MTs have been proposed to have a variety of cellular roles including metal ion homeostasis, detoxification and cytoprotection during the acute phase response(1, 2, 3, 4) . Consistent with a diverse array of cellular functions, MTs are transcriptionally induced by numerous agents including mitogens(5, 6) , cytokines(3, 4, 7) , and hormones(8) ; in many cases, the cis-acting elements that mediate the responses to inducers have been identified. However, the presence of such elements varies among MT genes in one family and among different species. For example, of the multiple members of the human MT gene family, only the hMT-IIA gene promoter has been shown to have a glucocorticoid-responsive element, and binding sites for AP1, AP2, and AP4, which mediate responses to activators of protein kinase C and protein kinase A pathways(9) . However, metals are the common inducer of MT genes, and all MT promoters from Drosophila to humans contain multiple copies of a semiconserved sequence that is responsible for induction by metals, called a metal-responsive element (MRE)(9) . MREs are 12-15- base pair sequences consisting of a highly conserved heptanucleotide core, TGC(A/G)CNC, and less conserved flanking nucleotides(9) .

To determine the cis-acting sequence requirement for metal induction of transcription in mammalian cells, previous studies have employed a variety of synthetic MREs fused to a minimal promoter(10, 11) . However, we are interested in MRE sequence contributions to transcription in the context of native promoter sequences, orientation and distance from the TATA box. The rainbow trout (Salmo gairdnerii) (t)MT-B gene provides a convenient tool for this purpose. The tMT-B promoter lacks the numerous basal and inducible cis-acting elements that interpose and surround the MREs of mammalian MT promoters(9) . Further, the tMT-B gene has only two MRE sequences within 250 base pairs of 5`-flanking sequence, whereas the analogous regions of mammalian MT promoters contain four to six MREs (9, 12) . Because of this apparent simplicity, we have employed site-directed mutagenesis of the tMT-B promoter to determine the contribution of each MRE to transcription activity.

By transient transfection of rainbow trout hepatoma (RTH-149) cells, we demonstrate here that the two MREs of the tMT-B proximal 5`-flanking sequence cooperate for a high level of metal induction. However, each MRE contributes differentially to basal and metal-induced promoter activity, and this variance is partly due to sequence differences within the MRE consensus. In mammalian cell lines, the differential contributions of each MRE for basal and metal-induced transcription are observed, but differences in the function of the tMT-B promoter suggest that additional or functionally distinct factors interact with the promoter in mammalian cells. With this in mind, the rainbow trout and mammalian nuclear factors that bind to the tMT-B promoter sequences vary in apparent molecular mass.

MATERIALS AND METHODS

Trout MT-B Promoter-Reporter Gene Constructs

Figure 1: Rainbow trout MT-B promoter deletions and MRE mutants. A schematic diagram of the tMT-B promoter is shown with the sequences of the wild type and mutated MRE sequences. Deletion and site-directed mutagenesis of the tMT-B promoter was performed as described in (13) and under ``Materials and Methods.'' The wild type and MRE mutant promoter fragments were fused to the reporter genes CAT and LUC in the vectors pMEV1R and pMEV35R, respectively.

All tMT-B wild type and mutant promoter fragments were subcloned into mammalian expression vectors pMEV1R and pMEV35R, which were constructed in our laboratory(15) . The vector pMEV1R contains a chloramphenicol acetyltransferase (CAT) reporter gene which was replaced with a firefly luciferase gene (LUC) to construct pMEV35R (15, 16, 17) . Plasmid DNA for transfection was purified using the Plasmid Maxi Purification Kit (Qiagen) according to the manufacturer's instructions.

Cell Culture and Transfection

The calcium phosphate precipitation method of Gorman et al.(16) was used for transfection of cells, and slight modifications to the procedure have been described previously in detail(12, 13, 15) . Cells were seeded at a density of 1 10 cells/cm and treated with 0.13 µg/cm test plasmid and an equal mass of the carrier plasmid pGEM or pUC13. After a 24-h recovery from glycerol shock, the transfected cells were treated with metal for 48 h for RTH-149 cells or 24 h for mouse L and HepG2 cells(12, 13, 20) . Zinc was chosen for metal induction since it is the most efficient inducer of the endogenous and transfected tMT-B gene in RTH-149 cells, eliciting the fastest, highest, and longest lived transcription response compared with cadmium and copper(12, 21) . At 100 µM ZnCl, there are minimal cytotoxic effects and growth inhibition without compromising the level of transcription activation(21) .

Reporter Gene Assays

The numerous constructs used in this study were assayed from two to six times with each reporter gene, and the results were found to be consistent. Basal and zinc-induced values of reporter gene activity were normalized by considering that the metal-induced activity of the -137 promoter fragment had a value of 100. After normalization, basal and metal-induced values for different trials of the same construct were averaged and are presented as units of relative activity. The standard deviations of trials for the different constructs under basal and metal-induced conditions were generally 5-15% of the average.

The LUC assay system allowed a larger range from minimum to maximum detection limits. Because of this increased sensitivity, quantitative values are presented for LUC for comparison with the qualitative results of a representative trial using CAT. However, the effects of mutations on metal fold induction using CAT assays are stated under ``Results'' from the average of three transfections.

Southwestern Blotting

The annealed double oligonucleotides and the tMT-B promoter PCR products were purified by elution from 10% nondenaturing polyacrylamide gel electrophoresis and end labeled to a specific activity of 10 cpm/µg as per accepted protocols(23) .

Trout liver extracts were prepared by the method of Gorski et al.(24) . Nuclear extracts from mouse L cells and HepG2 cells were prepared as described previously(25) . Nuclear proteins (50 µg) were fractionated using 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose(22) . Blots were soaked in binding buffer containing 10 mM Hepes, pH 7.9, 50 mM NaCl, 1 mM MgCl, 5 mM dithiothreitol, and 10% glycerol (26) along with 6 M guanidinium isothiocyanate for 10 min at room temperature and then transferred to 4 °C for all further steps. The denaturant was diluted 1:1 in binding buffer containing 100 µM ZnCl for 10 min and then diluted four more times in a similar fashion. After the final dilution step the blots were washed in binding buffer with zinc and then blocked 16 h in binding buffer with zinc and 5% nonfat milk. End-labeled probe was added to a concentration of 10 cpm/ml binding buffer with zinc and 0.25% nonfat milk for 7 h at 4 °C. The blots were washed twice in binding buffer with zinc for 10 min, air dried, and then exposed to x-ray film.

RESULTS

Promoter Fragments Containing MREa and MREb Both Contribute to Metal Induction of the tMT-B Promoter

Figure 2: CAT and LUC activity of tMT-B promoter deletions. RTH-149 cells were transfected and treated with metals as described under ``Materials and Methods.'' A representative CAT assay from untreated and metal treated cells is aligned with the relative LUC activity measured for each construct. Metal fold induction values are stated above each metal-induced LUC activity bar.

We chose to confirm the activities of the tMT-B deletions with a LUC reporter gene, since the 4.0-fold zinc induction was considerably lower than has been observed for the endogenous tMT-B gene in RTH-149 cells (27) . Using LUC, the basal levels of expression for -84 and -137 fragments were comparable, whereas metal-induced levels were increased significantly for -137 (Fig. 2, top panel). As a result, -84 displayed 3.5-fold metal induction, whereas -137 generated a 12.5-fold metal response. The greater metal inducibility observed using LUC is because this assay system displays a greater range from minimum to maximum detection limits compared with CAT. As a result of lower background expression from pMEV35R (LUC), measurement of basal activity is more easily determined. For example, the TATA box alone(-49) displays only 1% of metal-induced -137 activity, whereas this is an average of 15% using CAT. This decrease in basal expression values results in an increase in fold induction which more closely approaches that of the endogenous gene.

Results of both CAT and LUC assays suggested that both MREa and MREb are active for metal regulation since zinc inducibility increased with the addition of each MRE-containing promoter fragment to the TATA box. However, the fragment containing both MREs(-137) had metal-induced levels much greater than if MREb contributed to induction equivalent to MREa alone(-84). This suggested either that MREb makes larger contributions to metal induction than MREa, or MREa and MREb interact in a cooperative manner.

The tMT-B Promoter MREa and MREb Contribute Differentially to Basal and Metal-induced Activity

The results of both CAT and LUC assays of promoter activity in RTH-149 cells are combined in Fig. 3. Using LUC, the inactivating mutation of MREa (-60A) actually resulted in a substantial increase in metal induction from 12.5-fold for the wild type promoter to 16.7-fold. This was paralleled by the results of CAT assays which also revealed an increase in induction from 4.0 to 6.6-fold because of this mutation. Although this mutation was created to reduce the metal responsiveness of the promoter, the actual consequence was an extreme decrease in basal activity to the level of the TATA box(-49), increasing the difference between basal and induced activity. From this observation, MREa could be considered the dominant basal element in the -137 promoter fragment which concurs with the lack of other recognizable elements in the promoter(12) .

Figure 3: Activity of tMT-B promoter MRE mutants. A representative CAT assay is combined with the relative LUC activity of the wild type tMT-B promoter(-137) and MRE mutants in RTH-149 cells averaged from three trials. The metal fold induction from the ratio of zinc-induced activity to basal activity is stated above each zinc-induced LUC activity bar.

The analogous mutation of MREb (-91T) was much less devastating to overall promoter activity, decreasing the basal level by only 15%, but reducing induction to 9.5-fold using LUC. Similarly, the fold induction of the analogous CAT construct also decreased compared with the native promoter. This confirms that the MREb sequence also contributes to the metal responsiveness of the tMT-B promoter.

The double mutant, -137/-60A/-91T, displayed the same low basal activity expected from the result of the single MREa (-60A) mutation. In addition, zinc-induced levels measured by LUC were reduced to only 4% of the wild type promoter. However, in spite of mutation of both MREs, this construct still exhibited 3.1-fold induction with LUC and 1.9-fold induction with CAT. From this result, the single point mutations intended to inactivate MRE function still allow metal induction in the context of the tMT-B promoter. This contrasts previous studies with synthetic promoters where mutation at this first MRE core nucleotide completely inactivated MRE function(10, 11) .

Since the point mutated MREs were still partially active, the independent and cooperative contributions of the MREs to tMT-B promoter function were not well defined. Additional mutants were constructed, -137/dMREa and -137/dMREb, where each MRE sequence was substituted at numerous nucleotides so that it was unrecognizable as an MRE while maintaining the position of the second MRE and other possibly important sequences (Fig. 1).

Substitution of MREa (-137/dMREa) was devastating to the basal activity of the promoter, expected from the result of -137/-60A mutant promoter (Fig. 3). However, zinc-induced levels also decreased to nearly undetectable levels. Without MREa, 2.1-fold induction of promoter activity was observed with LUC, whereas no inducibility was observed with CAT assays.

The substitution of the MREb sequence was not as devastating to promoter activity compared with substitution of MREa since -137/dMREb-LUC still displayed activity well above the TATA box alone (-49) and an induction of 3.7-fold. For CAT, basal level was the same as the native -137 promoter fragment, but metal-induced activity was reduced, resulting in a metal induction of 1.3-fold. For both assay systems, the metal fold induction of -137/dMREb is comparable to that of -84 which also contains only MREa.

The use of these substitutions of MREa and MREb were more revealing than the single point mutants in regard to the role of each MRE in tMT-B promoter activity. MREa appears to be able to function in an independent manner since substitution of MREb still allows basal activity above the TATA box alone and elicits a significant response to metals. However, substitution of MREa devastates basal promoter activity and allows only nominal metal induction, indicating that MREb cannot function efficiently independent of MREa. Both MREs must cooperate for a response to metals since the sum of the fold inductions generated by each MRE when the other was substituted does not equal that of the native promoter.

Differential MRE Function Is Partially Determined by Sequence

We considered that the observed differential function of MREa and MREb for basal and metal-induced tMT-B activity could be due to position relative to the TATA box, orientation, or subtle sequence differences between the MREs. MREa and MREb differ by only one nucleotide (underlined), TTTGCACNCGG, which is -55A in MREa and -96G (C in forward orientation) in MREb (Fig. 1). This position is the least conserved nucleotide of the MRE consensus since all 4 bases have been found to occupy this position in natural MREs (9) . To determine if this nucleotide was responsible for differential basal and metal-induced MRE activity, reciprocal point mutants were made to alter MREa to the MREb core sequence, -137/-55C, and MREb to the MREa core sequence, -137/-96T (Fig. 1). If this nucleotide difference was responsible for the observed basal activity of MREa, then -55C should reduce the basal activity of MREa, and therefore the promoter, while -96T should increase the basal contributions of MREb.

The effects of these mutations on tMT-B promoter activity are shown in Fig. 4. For both CAT and LUC, mutation of MREa to TGCACCC (-137/-55C) caused a significant decrease in basal promoter activity but had little effect on metal-induced activity. As a result, the induction was increased 12.5-17.5-fold for LUC, and 4.0-11.2-fold for CAT. The opposite mutation of MREb to the MREa sequence (-137/-96T) increased basal activity above that of the native promoter. For LUC, the metal-induced levels were also increased nearly 1.5-fold, whereas for the CAT assays, metal-induced levels were not affected significantly. As a result of the increased basal level, metal inducibility was decreased compared with the native promoter, from 12.5- to 10.2-fold for LUC and from 4.0- to 3.3-fold for CAT.

Figure 4: Effect of MRE core sequence on basal activity. See Fig. 3legend.

The effect of MRE core sequence on basal promoter activity was investigated further using the MRE reverse mutations, -55C and -96T, in concert with the inactivating point mutants of the opposite MRE. The devastating effect of the MREa mutation (-60A) on basal activity was partially rescued by altering MREb to an MREa sequence for the construct, -137/-96T/-60A (compare Fig. 3and Fig. 4). This was a 2.6-fold increase in basal expression for LUC compared with the -137/-60A construct and a smaller but visible increase for CAT. Conversely, the basal activity of -137/-55C/-91T was decreased compared with -137/-91T ( Fig. 3and Fig. 4).

All of the above constructs served to show that the N position of the MRE core sequence is an important determinant of MRE contributions to basal activity. When both MREs have the sequence of MREa, the basal activity of the promoter is increased; the opposite is true when both MREs have the MREb sequence. This is consistent with the results of the MRE point mutants which suggest that MREa is an important contributor to basal level expression. However, the mutation of MREb to an MREa sequence could not confer independent activity on MREb, since the -96T mutation did not completely rescue the basal activity of the -60A mutant. Because of this observation, it was important to determine if the orientation and position of the MREs were also important. The double mutant, -137/-96T/-55C, is the reciprocal of the tMT-B promoter with the MREa sequence in MREb position and vice versa. This construct had the highest general activity of all of the mutant promoters. For LUC, both basal and metal-induced levels nearly doubled, but the resulting 12.3-fold metal induction was similar to that of the native promoter (12.5-fold). The homologous CAT construct also had metal induction (3.9-fold) comparable to that of the wild type promoter (4.0-fold), although the increase in general promoter activity was not as large as observed with LUC assays. Since fold induction is conserved with this reversal of MRE core sequence, the cooperative interactions between the two MRE core sequences for metal inducibility are not affected. However, general promoter activity is increased compared with the native promoter so that position, orientation, and/or flanking sequences are also important for basal contributions to the promoter.

MREa Is a Strong Basal Element in Mammalian Cells, but the Metal Responsiveness of the tMT-B Promoter Is Reduced

Previously, it has been observed that tMT-B -84-CAT is active in mouse L cells(13) . To determine if the differential basal and metal-responsive activities of MREa and MREb are conserved in mammals, we have tested the most important tMT-B promoter-LUC constructs in mouse L cells. As with RTH-149 cells, tMREa also acts independently in mouse L cells, with -84-LUC eliciting a 3.7-fold induction with zinc (Fig. 5A). However, adding sequences from -84 to -137 lowers general promoter activity. Further, the resultant induction is only 4.6-fold for -137 (Fig. 5A), which is much lower than the 12.5-fold induction observed in trout cells. The less significant metal induction in mouse L cells could be caused by the high basal activity of the tMT-B promoter. In RTH-149 cells, -84 increased basal activity 6.5-fold over the TATA box(-49) alone; this was observed as an 113-fold increase in mouse L cells.

Figure 5: Basal and metal-induced activity of tMT-B promoter in mammalian cells. Selected tMT-B promoter-LUC fusions were transfected into mouse L cells (panel A) and human HepG2 cells (panel B) and treated as stated in under ``Materials and Methods.'' The relative LUC activity is the mean of two separate trials.

To determine if the high basal level involved MREa, the inactivating mutation -60A was tested. This mutation decreased basal activity more than metal-induced levels, as in trout cells, increasing induction from 4.6- to 18.1-fold. The analogous MREb mutant, -137/-91T, actually increased basal activity but did decrease metal-induced expression, reducing induction to 3.4-fold, similar to the 3.7-fold induction of MREa alone(-84), confirming that MREb does make some contributions to metal induction. Finally, the double mutant 137/-60A/-91T displayed the same low basal level as the -60A mutant and was still 3.0-fold induced by zinc so that the single point mutations of the MREs do not completely inactivate metal inducibility as observed in trout cells.

The tMT-B promoter was still able to respond 3.9-fold to metals with substitution of the MREa sequence (-137/dMREa), although the basal level was devastated. In this sense, MREb was able to act in an independent manner for metal induction. When MREb was substituted, the promoter retained the basal level of the promoter, through MREa, but inducibility was reduced to 2.4-fold.

Although the basal contributions of MREa were observed in mouse L cells, as in RTH-149 cells, there were important differences in expression of tMT-B promoter fragments. The addition of promoter regions from -84 to -137 appeared to inhibit basal activity, and the metal fold induction of the promoter was reduced. Also, the mutation of MREb (-91T) noticeably increased basal activity. These results were unanticipated and merited further testing of these constructs in another mammalian cell line.

Human HepG2 cells were chosen because this cell line has been useful for understanding human MT gene expression(19, 20, 29, 30) . As in mouse L cells, the tMT-B promoter fragments -84 and -137 displayed high basal activity and low metal inducibility, with both fragments allowing only a 2.5-fold response to zinc, so the addition of MREb did not increase inducibility (Fig. 5B). Again, a decrease in general promoter activity was observed with the addition of sequences from -84 to -137. Compared with mouse L cells, this was much more obvious with promoter activity reduced almost in half.

As observed for trout and mouse cell lines, the -137/-60A mutant exhibited drastically reduced basal activity, which increased induction from 2.5- to 7.1-fold (Fig. 5B). However, mutation of MREb, -137/-91T, also increased induction to 3.8-fold by increasing metal-induced expression without affecting basal activity (Fig. 5B). Finally, the double MRE mutant -137/-91T/-60A still had significant induction (1.9-fold) compared with the native promoter (2.5-fold) (Fig. 5B).

The behavior of the tMT-B promoter constructs in mammalian cell lines indicates that the tMT-B MREa also contributes to basal activity in mammals. However, the cooperative interactions of MREa and MREb are reduced so that the metal fold induction of the promoter is substantially less than that observed in trout cells. In addition, the more distal promoter fragment from -84 to -137 appears to repress promoter activity, which may be responsible for the lesser contributions by MREb in this region. Since the point mutation of MREb (-91T) does increase basal activity of the promoter in mouse L cells and metal-induced levels in HepG2 cells, it seems unlikely that this repression could only be caused by context effects from the nearby vector sequences.

Proteins Interact with the tMT-B Promoter Sequences in Trout Liver, Mouse L Cell, and HepG2 Cell Nuclear Extracts

Figure 6: Southwestern blotting analysis. Nuclear extracts from rainbow trout liver (t), mouse L cells (m), and HepG2 cells (h) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose; blots were treated as described under ``Materials and Methods.'' Panel A, the wild type (W.T.) tMT-B promoter region (-137 to -30) was used as a probe and identified single proteins in each of the nuclear extracts. The double MRE mutant tMT-B promoter fragment (-91T/-60A) was incapable of binding to the same proteins, and the inclusion of o-phenanthroline instead of zinc during binding abolished binding of the wild type probe (W.T./-Zn). Panel B, a tMREa oligonucleotide probe (W.T.) identified the same proteins as the tMT-B promoter while binding was abolished for the point mutated tMREa (-60A) or for the wild type tMREa in the presence of o-phenanthroline and absence of zinc (W.T./-Zn).

To confirm that the MRE sequences were responsible for the interaction of the promoter with the nuclear proteins in each extract, a double-stranded tMREa oligonucleotide was used as probe (Fig. 6B). In each extract, tMREa identified the same proteins as the whole tMT-B promoter fragment. The tMREa -60A point mutant did not bind to the proteins, and, as with the whole promoter, the absence of zinc because of chelation by o-phenanthroline abolished binding (Fig. 6B).

Although this method cannot identify proteins that bind to the tMT-B promoter as a heterogeneous complex, it does indicate that the only independent protein-tMT-B promoter interactions are MRE-specific and zinc-dependent. It also does not identify any MRE-independent mammalian proteins that might be responsible for the decreased function of the tMT-B promoter MREb region. It is interesting that the proteins bound by the tMT-B promoter and MREa probe in trout, mouse, and human extracts display appreciable differences in size even though the MRE consensus sequence is highly conserved from trout to mammals. It is possible that the DNA binding domain is highly conserved, but other functional domains are divergent, which could be responsible for the differences in tMT-B promoter activity in RTH-149 cells compared with mouse L and HepG2 cells.

DISCUSSION

Using transfection analysis, we have determined that the two MRE sequences present in the tMT-B promoter cooperate for a significant response to zinc in a rainbow trout hepatoma cell line. We also have noted that tMREa is zinc-responsive without cooperative interactions with a second MRE. This is an unusual characteristic, which has been observed only with the mouse MT-I MREd sequence(11) . From additional results not presented here, independence is not conferred on tMREb if mutated to the tMREa sequence (-137/-96T/dMREa). ()Since distance from the TATA box has been shown to affect MRE function in synthetic promoters(10) , the independent activity of tMREa may be a consequence of close proximity to the TATA box, rather than sequence.

We previously have shown that basal MT levels are significant in rainbow trout tissues and primary cell culture(27, 31) . From our results, there are no additional basal elements other than MREa in the proximal tMT-B 5`-flanking sequence. In past discussions, MREs have been considered only as inducible elements because mammalian MT promoters contain numerous other potential basal elements (9, 28, 32, 33, 34) and MREs generally are not protected in vivo prior to metal induction(28, 32) . Nevertheless, tMREa is also a basal element in mammalian cell lines, so the basal activity of MRE-binding factors is conserved from trout to mammals. We detect only one protein in each of the trout and mammalian nuclear extracts which binds tMREa, but multiple MRE specific proteins previously have been detected within one cell line or species which could have distinct functions for basal and metal-induced MT expression(13, 26, 35, 36, 37, 38, 39, 40, 41) . However, the cloned mouse MRE-binding factor, MTF-1, is required for both basal and metal-induced MT gene expression, supporting a role for MREs in basal MT transcription(26, 42) . The zinc-dependent mouse and human proteins that we detect using the tMREa sequence are similar in apparent molecular mass to mouse and human MTF homologues(26, 41) , which are zinc finger proteins, so it is possible that these are the factors responsible for tMREa basal activity in mammalian cell lines. In fact, the mouse MTF-1 cDNA was cloned from an expression library by affinity to an MRE with the same core sequence as tMREa(26) .

The basal properties of tMREa, in contrast to tMREb, are partially due to a nucleotide difference of A instead of C at the variable position of the core sequence, TGC(G/A)CNC. Searle et al.(10) also have observed a substantial increase in basal activity of a synthetic MRE dimer when A is substituted for C at this position. An A rather than G nucleotide at the fourth position of the TGC(G/A)CNC core, as in tMREa, also raises basal activity of a synthetic MRE sequence (10) as well as increasing binding of a zinc-dependent factor from rat liver nuclear extracts(36) . Therefore, the functional effects of an A at these two variable positions of the MRE core may be a reflection of increased affinity of MRE-binding factor(s), and the tMT-B MREa could be an ideal core sequence for factor binding. If only a low concentration of the factor is functional in the cell, binding would be limited to high affinity MREs, such as the tMREa sequence, for basal activity. In the presence of metal, higher concentrations of MRE-binding factor could be available to bind lower affinity sites through cooperative interactions with the constitutively bound MRE(s), eliciting a metal response that is significantly higher than basal expression. There is sufficient variability among MRE sequences in one MT promoter, both within the core as well as flanking sequences, to allow two levels of MRE-dependent transcription(9) . Although the tMREa sequence is common in trout MT promoters, ()of the mammalian MT-I isoform genes we have examined, only mouse MREe, human MT-IF MREc, and rat MREf have this core sequence(10, 15, 32) . However, the activity of the proximal basal level enhancer of the human (h)MT-IIA is abolished without a TGCACAC sequence in opposite orientation at its 3`-boundary, suggesting that this MRE sequence is required for hMT-IIA basal activity(33) . This same tMREa core sequence is also found in the distal basal level enhancer(33) . Other human MT promoters with less significant basal level activity compared with hMT-IIA may have less high affinity sites because their numerous MREs, in cooperation with other non-MRE elements, would result in a loss of metal regulation.

Although both trout MREs also are active in mammalian cell lines, the metal responsiveness of the promoter is reduced. Since the fold induction accorded by tMREa alone(-84) is nearly as significant in mammalian cells as in RTH-149 cells, the reduced metal responsiveness of the whole promoter(-137) fragment could be caused by smaller contributions by MREb. We do observe some inhibition of tMT-B activity in mammalian cells by sequences in the MREb region (from -84 to -137) which is partially relieved by point mutation of MREb. However, we have found no previous evidence to support an additional role for MREs as repressors of MT transcription, although the MRE affinity-purified HeLa cell factor MRE-BP has been suggested to be a negative regulatory factor(38) . An alternative explanation for the decreased fold induction of the trout promoter in mammalian cell lines is that the MRE organization may favor the cooperative function of the homologous trout MRE-binding factors, whereas the heterologous mammalian factors are less able to accomplish synergistic activation of transcription even though they bind and activate from single MREs. Although the MRE consensus sequence is conserved from trout to mammals, MT promoter organization is not. Mammalian MT promoters contain four to six MRE sequences, whereas the two MREs of the tMT-B promoter can elicit a comparable metal response(9) . In this sense, the lesser ability of mammalian metalloregulatory factors to cooperate for activation of the tMT-B promoter may be a reflection of species-specific differences in MT promoter organization and the MRE-binding factor domains responsible for the synergism of MREs.

Our results presented in this study indicate that MREs can function as basal level elements in addition to their role in metal induction. Combined with a recent report that the mouse MRE-binding factor MTF-1 is absolutely required for mouse MT-I gene basal expression, it is possible that the cis-acting elements present in MT promoters are organized such that they are inactive without the physical effects of MRE-binding factors. In this sense, a key to understanding MT gene transcription regulation will be to characterize fully the physical effects of factor interactions with MREs of MT promoters and understand the mechanisms that are involved in the synergism among MREs and other cis-acting elements. In the future, a comparison of the functional domains of MRE-binding factors from divergent species, such as trout and mammals, would be one approach to understanding MRE function.

FOOTNOTES

*

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

§

Supported by a studentship from the Alberta Heritage Foundation for Medical Research.

¶

To whom correspondence should be addressed: Dept. of Biology, University of Calgary, 2500 University Dr. N. W., Calgary, Alberta, Canada T2N 1N4. Tel.: 403-220-5556; Fax: 403-289-9311.

()

The abbreviations used are: MT(s), metallothionein(s); MRE, metal-responsive element; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; LUC, firefly luciferase; tMRE, rainbow trout MT-B gene MRE.

()

S. L.-A. Samson, unpublished observation.

()

S. Scheiman, unpublished observation.

ACKNOWLEDGEMENTS

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