MT Promoter Research Articles

PF1070A, a Novel and Potent Inducer of the Synthesis of Metallothionein

Using mouse Ltk(-) cells (L13-17 cells) that had been transfected with a plasmid in which the lacZ gene had been ligated downstream of 1.4 kbp of the sequence of the promoter of the mouse gene for metallothionein-I (MT-I) as a reporter gene, we examined 268 organic compounds for the ability to activate this promoter. We found that PF1070A, an antibiotic produced by Humicola sp., efficiently activated the MT promoter and caused marked enhancement of beta-galactosidase activity in L13-17 cells. The extent of activation by PF1070A was almost equivalent to that by of zinc ions, the most effective known inducer of the synthesis of MT. PF1070A also caused marked elevation of the levels of the mRNA for MT and of MT itself in L13-17 cells. A similar result was obtained in human HeLa-S3 cells. When PF1070A was added to the culture medium simultaneously with cadmium ion or dexamethasone, the level of expression of the reporter gene was markedly elevated, compared to the level of expression induced by each agent independently. The effect of PF1070A was reduced considerably by deletion of nucleotides at positions -150 and -149 from the site of initiation of transcription in the promoter region of the MT gene and also by deletion of the seven bases located at positions -49 to -43. Since no known cis element was found in these two regions, PF1070A might be a new type of inducer of MT synthesis that promotes expression of the gene for MT via a mechanism completely different from those exploited by other known agents. These results also suggest the presence of a system for control of transcription of the gene for MT that has not previously been recognized. Both cadmium ions and bismuth ions induce the synthesis of MT by acting on the metal response element (MRE). Bismuth ions had no significant effect on the promoter activity that had already reached a maximum level in response to treatment with the optimal concentration of cadmium ion. By contrast, PF1070A further and markedly increased the promoter activity. This result suggests that it is possible to increase the concentration of MT in tissue using PF1070A as an inducer even in cases where the MRE-mediated activation of the MT promoter has already been induced by the accumulation of cadmium, as is the case in a clinical setting. PF1070A may prove to be an excellent inducer of MT synthesis that is effective and clinically applicable. Moreover, use of PF1070A in combination with salts of heavy metals might be useful in controlling expression of a transfected gene that is regulated by the MT promoter since PF1070A can activate the MT promoter to an extent that cannot be achieved with heavy metal ions alone, when PF1070A is used in combination with zinc ions at a concentration of the latter considerably below the toxic level.

Transfer of an ovine metallothionein-ovine growth hormone fusion gene into swine.

An ovine metallothionein-1a (oMT1a)-ovine growth hormone (oGH) fusion gene was microinjected into 400 pig zygotes, the zygotes were transferred into recipient females, and 15 founder transgenic pigs were born. Of 12 transgenic pigs assayed, five expressed high levels of oGH (> 900 ng/mL plasma), one expressed low levels of oGH (10 to 30 ng/mL), and six did not express oGH. Dietary supplementation with 2,000 ppm of zinc for 6 d induced a 20-fold increase in plasma oGH in the transgenic pig with low expression but did not induce expression in the six transgenic pigs with no constitutive oGH expression. The average daily gain of five transgenic pigs with elevated oGH was similar to that of non-transgenic littermates during a 9-wk feeding trial (P = .52). The liver, kidney, adrenal, and thyroid weights were all significantly heavier for the oGH-expressing transgenic pigs than for non-transgenic littermates. Total carcass fat, longissimus muscle fat, subcutaneous backfat thickness, and loin eye area were lower and carcass protein and water content and beta R fiber area of longissimus muscle were higher in the transgenic pigs with elevated oGH than in their littermate controls (P < .05 for each). The data indicate that even though the oMT1a promoter was more inducible by zinc than was previously reported for the mouse MT promoter in swine, the former provided a higher level of oGH expression than the mouse MT promoter.

Activation of the Chicken Metallothionein Promoter by Metals and Oxidative Stress in Cultured Cells and Transgenic Mice

Cis-acting elements in the chicken metallothionein promoter were tested for their ability to direct responses of reporter genes to metal ions and oxidative stress in transfected mouse cells and in transgenic mice. In addition, protein interactions with the promoter were analyzed by the electrophoretic mobility shift assay. In transient transfection assays and in transgenic mice, 107-bp of the chicken MT promoter was sufficient to direct responses to Zn. This promoter region also directed response to oxidative stress in transfected cells and transgenic mice, but in transgenic mice, maximal responsiveness to oxidative stress apparently involved other elements in the proximal promoter region (307-bp). The proximal 200-bp of the promoter contains sequences homologous to a metal response element (−47-bp), Sp1 binding sites (−70-bp and −161-bp), and an antioxidant response element (−189-bp). Electrophoretic mobility shift assay demonstrated that metal response element binding activity was low in control Hepa cell nuclear extracts, but was induced 6-fold after 45 min of H2O2 treatment. In contrast, Sp1 binding remained unchanged, and no evidence for specific binding to the core antioxidant response element consensus sequence was obtained. These studies demonstrate that cis-acting elements mediating induction of metallothionein gene expression by metals and oxidative stress are present in the chicken metallothionein promoter and suggest a role for increased binding of the transcription factor MTF-1 to the metal response element(s).

Regulation of metallothionein genes by the ACE1 and AMT1 transcription factors

The AMT1 metalloregulatory trans-acting factor from Candida glabrata was found to functionally mimic the ACE1 metalloregulatory trans-acting factor from Saccharomyces cerevisiae in the copper-induced expression of the chromosomal S. cerevisiae metallothionein gene. Plasmid constructs with promoters of various metal-inducible genes fused to the bacterial beta-galactosidase (lacZ) reporter gene were used in S. cerevisiae to evaluate the roles of ACE1 and AMT1 in mediating metal-stimulated expression. Promoters from the S. cerevisiae CUP1 gene and Cu,Zn-superoxide dismutase (SOD1) and from the C. glabrata MT genes MTI, MTIIa, and MTIIb were used. The ACE1 factor was effective in the metalloregulation of the two S. cerevisiae promoters, CUP1 and SOD1, but of only one C. glabrata promoter, MTI. AMT1 was found to be effective in the metalloregulation of all three C. glabrata MT promoters and the two S. cerevisiae promoters tested. The regulation mediated by both ACE1 and AMT1 was copper-dependent and copper-specific. Episomally expressed SWI5, a distinct trans-acting factor of S. cerevisiae, enhanced only the basal expression from promoters. The SWI5 enhancement was not metal dependent. In conclusion, AMT1 and ACE1 are functionally homologous in metal-specific regulation, AMT1 appears to be more promiscuous than ACE1 in this function.

Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line.

A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.

Synergistic induction of promoters containing metal- and glucocorticoid-responsive elements.

We have shown that it is possible to synergistically activate gene transcription when several glucocorticoid responsive elements (GREs) and metal responsive elements (MREs) that coexist within the same promoter are induced simultaneously. To demonstrate this, additional GREs were introduced into a human metallothionein IIA (hMT-IIA) promoter in which some constitutive elements had been deleted. The transcriptional strength and inducibility of the modified hMT-IIA promoters were studied in transient expression experiments using the CAT gene as a reporter. As a result of synergistic activation of transcription by CdCl2 and dexamethasone, the induced expression levels of the modified promoters were significantly higher than those obtained with wild-type hMT-IIA. Since the increase in inducible expression was not accompanied by a concominant increase in basal levels, the inducibility of the modified MT promoters was up to 6-fold higher. The degree of transcriptional synergism was shown to depend on the position and the number of GREs introduced. Thus, the engineering of synthetic promoters that include both GREs and MREs should offer the opportunity to develop a new series of improved inducible mammalian expression vectors.

Characterization of Chinese hamster ovary cells stably transformed by a plasmid with an inducible APRT gene

A plasmid was constructed by fusion of a selectable mammalian gene, hamster adenine phosphoribosyltransferase ( APRT), to the Zn 2+-inducible sheep metallothionein I (MT I) promoter. This plasmid was used to produce stable Chinese hamster ovary (CHO) cell transformants by electroporation to study the effects of induced gene expression on DNA-mediated transformation. The sheep MT Ia promoter was chosen for these experiments because it regulates gene expression differently than murine MT promoters, exhibiting low basal levels of gene expression in uninduced conditions. We have shown that in the absence of Zn 2+, there is very low expression of a sheep MT I- APRT fusion gene in stable CHO cells transformants; induction of APRT mRNA and enzyme activity by Zn 2+ produced a “threshold” response, from low basal levels to high induced levels, in Zn 2+ responsive stable transformant clones. In electroporation experiments, transformation frequencies were unaffected by Zn 2+ treatments during a preselection period, but the presence of Zn 2+ during selection increased the recovery of stable transformant clones 8- to 10-fold. All stable transformants analyzed displayed Zn 2+-inducible APRT enzyme activity. Our results indicate that stable mammalian cell transformants with inducible genes under regulation of the sheep MT I promoter should be useful, because of low basal and high induced expression, for studies in which modulation of transcriptional activity is required.

Transformation of mouse BALB 3T3 cells by enterobacterial plasmid misrepair gene mucAB.

The enterobacterial plasmid misrepair gene mucAB, ligated to the metal-inducible mammalian MT-1 promoter, was introduced into the genome of mouse BALB 3T3 cells. In the presence of zinc ions, MucA but not MucB protein was produced, and the whole-cell population of each mucAB+ clone started to show the transformation phenotype in a few days. Foci appeared in the transformed cell population after 4 weeks, and cells from the foci produced tumors in nude mice, indicating malignant transformation by the mucA product. Growth of mucAB+ cells was stimulated by zinc-induced expression of mucA. The transformation phenotype was reversed by removing zinc ions from the culture, indicating that the transformation was due not to MucA-mediated mutation in the mouse genome but to the direct transforming activity of MucA protein.

Transformation of Mouse BALB 3T3 Cells by Enterobacterial Plasmid Misrepair Gene mucAB

The enterobacterial plasmid misrepair gene mucAB, ligated to the metal-inducible mammalian MT-1 promoter, was introduced into the genome of mouse BALB 3T3 cells. In the presence of zinc ions, MucA but not MucB protein was produced, and the whole-cell population of each mucAB+ clone started to show the transformation phenotype in a few days. Foci appeared in the transformed cell population after 4 weeks, and cells from the foci produced tumors in nude mice, indicating malignant transformation by the mucA product. Growth of mucAB+ cells was stimulated by zinc-induced expression of mucA. The transformation phenotype was reversed by removing zinc ions from the culture, indicating that the transformation was due not to MucA-mediated mutation in the mouse genome but to the direct transforming activity of MucA protein.

Regulated expression of the human mutant ras gene after transfection of BALB/c mouse embryo fibroblast cells.

Four continuous cell lines constructed by transfecting BALB/c mouse fibroblast cells with an expression system that has the mutant c-Ha-ras gene under control of a truncated version of the mouse metallothionein-1 (mt-1) promoter were characterized for zinc-induced phenotype switching. These cells were selected for transformation in the presence of zinc, a known inducer of the mt-1 promoter. When the transfected cells were grown in medium depleted of zinc, there was a dramatic reduction in their soft agar cloning efficiency. Adding zinc back to the medium restored the transformed phenotype in a dose-dependent manner. Analysis of the intracellular p21 levels of induced and uninduced cells showed that zinc was modulating the expression of the transfected ras gene. In vivo studies done with syngeneic mice showed that zinc-induced cells were tumorigenic and formed metastatic lesions in the lungs of the inoculated animals.

Expression of Human Uterine Tissue-Type Plasminogen Activator in Mouse Cells Using BPV Vectors

Human tissue-type plasminogen activator (t-PA) cDNA was cloned from uterine tissue and engineered in expression vectors for production in mouse C127 cells. The vectors consisted of the bovine papilloma virus-1 (BPV-1) genome and t-PA transcriptional unit with a mouse metallothionein (MT-1) promoter at the 5' end and MT-1 genomic sequences or SV40 early introns and polyadenylation signals at the 3' end. Analysis of the expression vectors transfected into cells revealed that t-PA is expressed 100- to 200-fold more with an intronless vector utilizing the SV40 polyadenylation signal than with other, intron-containing vectors. RNA analysis of stable cell lines indicated that t-PA expression levels correlated with mRNA abundance. DNA copy number and transcriptional rate of the MT-1 promoter remained constant in cell lines transformed by different BPV expression vectors. Uterine t-PA produced by recombinant DNA means was enzymatically active and similar in properties to Bowes melanoma t-PA.

A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene.

To define DNA sequences involved in mouse metallothionein-I (MT-I) gene promoter function and metal regulation, we fused the 5' flanking sequences of the MT-I gene to the coding sequences of a viral thymidine kinase (TK) gene. A series of 5' deletion, 3' deletion, linker-scanning, and internal deletion mutants of the MT-I promoter was constructed and assayed by microinjection into mouse eggs. The results indicate that at least two related promoter elements can confer some metal regulation independently. Those mutations that had the most severe effect on regulation impinge on a 12-base-pair conserved sequence that is repeated several times within the mouse MT-I and other MT promoters. To test the regulatory function of this sequence, it was synthesized as a pair of complementary oligonucleotides and inserted into the promoter of the TK gene. A single insertion of this sequence conferred limited metal regulation onto the TK promoter, whereas a construct with two separate inserts was regulated as efficiently as the MT-I promoter.