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The metalloregulatory functions of metal-response element-binding transcription factor-1 (MTF-1) have been mapped, in part, to its six highly conserved zinc fingers. Here we examined the ability of zinc finger deletion mutants of mouse MTF-1 to regulate the endogenous metallothionein-I (MT-I) gene in cells lacking endogenous MTF-1. MTF-1 knockout mouse embryo fibroblasts were transfected with expression vectors for FLAGtagged MTF-1 (MTF-1 flag ) or finger deletion mutants of MTF-1 flag and then assayed for metal induction of MT-I gene expression, nuclear translocation, and in vitro DNA-binding activity of MTF-1 and its stable association with the endogenous chromosomal MT-I promoter. Intact MTF-1 flag restored metal responsiveness of the MT-I gene, underwent nuclear translocation, displayed increased in vitro DNA binding in response to zinc and less so to cadmium, and rapidly formed a stable complex with the MT-I promoter chromatin in response to both of these metals. In contrast, although deletion of finger 1, fingers 5 and 6, or finger 6 only had variable effects on the nuclear localization and in vitro DNA-binding activity of MTF-1, each of these finger-deletion mutants severely attenuated metal-induced MTF-1 binding to the MT-I promoter chromatin and activation of the endogenous MT-I gene. These results demonstrated that the metal-induced recruitment of MTF-1 to the MT-I promoter is a rate-limiting step in its metalloregulatory function and that an intact zinc finger domain is required for this recruitment. During the course of these studies, it was discovered that mouse MTF-1 is polymorphic. The impact of these polymorphisms on MTF-1 metalloregulatory functions is discussed.Metal-response element (MRE) 1 -binding transcription factor-1 (MTF-1) is an essential metalloregulatory transcription factor that coordinates the expression of genes involved in zinc homeostasis and protection against metal toxicity and oxidative stresses. These include, but are not limited to, metallothionein (MT) (1), zinc transporter-1 (2), and ␥-glutamylcysteine synthetase heavy chain genes (3). However, the mechanisms by which MTF-1 activates gene expression in response to metals are not well understood.Treatment of mammalian cells with zinc in vivo promotes rapid nuclear translocation of MTF-1 (4, 5) and causes a dramatic increase in MTF-1 DNA-binding activity measured in vitro (6 -8) concomitant with the occupancy of MREs in the MT-I promoter in vivo (6, 9, 10) and the activation of MT-I gene expression (11). Direct interactions between zinc and the zinc finger domain of MTF-1 regulate its DNA-binding activity (7). This suggests a model in which MTF-1 senses free zinc, adopts a reversible DNA-binding conformation, moves to the nucleus, and activates gene expression by associating with the promoter (12, 13). However, cadmium, a more potent inducer of MT-I gene expression than zinc, is less effective than zinc at driving MTF-1 to the nucleus (4, 5), has little effect on the DNAbinding activity of MTF-1 in vitro (8), yet requires MT...

The in vivo association of transcription factors with the metallothionein-I promoter was examined using chromatin immunoprecipitation (ChIP) assays. The results demonstrated that c-fos is rapidly recruited along with the metal response element-binding transcription factor-1 (MTF-1) to this promoter in response to zinc or cadmium, and that this recruitment is reversed in the visceral yolk sac by a zinc-deficient diet in vivo, and in cultured cells after lowering the zinc concentration in the medium or during prolonged zinc exposure. In contrast, the interactions of c-jun, USF-1, USF-2 and Sp1 with this promoter are metal-independent. Studies of knockout cells revealed that the recruitment of c-fos to the MT-I promoter requires MTF-1, but that c-fos is not essential for recruitment of MTF-1 and metal-induction of MT-I gene expression. Studies of Hepa cells stably-transfected with reporter genes driven by the MT-I promoter suggested two in vivo binding sites for USF-1 and -2. In contrast, Sp1 was apparently associated with a single binding site (upstream of -153 bp). In addition, maximal recruitment of c-fos by metals required sequences and/or other proteins that interact upstream of -153 bp. In summary, these studies extend our understanding of the complexity and dynamics of the transcription factor complex that forms at the MT-I promoter in vivo in response to metals.

The zinc finger protein, metal response element-binding transcription factor-1 (MTF-1) regulates the expression of genes in response to metal ions and oxidative stress. The precise mechanisms by which this occurs are not understood. To further examine this problem, mouse MTF-1 was expressed in Saccharomyces cerevisiae and tested for the ability to activate metal response element-driven reporter gene expression. Zinc was an effective inducer of reporter gene expression. In general, the magnitude of zinc induction was dependent on the concentration of zinc in the culture medium, but independent of the amount of MTF-1 expression. Zinc induction also occurred with either integrated or episomal reporter plasmids containing the native mouse metallothionein-I proximal promoter. Deletion of fingers 5 and 6 of MTF-1, which function in a zinc-dependent manner to stabilize the DNA-binding activity of the protein in vitro, did not diminish the zinc induction of either episomal or integrated promoters. However, a Gal4 DNA-binding domain- MTF-1 fusion protein, which binds constitutively to the Gal4-responsive promoter, was not zinc inducible but caused constitutive activation of reporter gene expression. This suggests that zinc activation of the DNA-binding activity of MTF-1 is the rate limiting step in its metalloregulatory function in yeast. In contrast, MTF-1 was not responsive to either cadmium or hydrogen peroxide, suggesting that distinct co-activators or signal transduction cascades not found in yeast are required to mediate MTF-1 activation of gene expression by this toxic metal and by oxidative stress.

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