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- Metal-Responsive Transcription Factors That Regulate Iron, Zinc, and Copper Homeostasis in Eukaryotic Cells

To a major extent, zinc homeostasis generally parallels copper homeostasis in that both posttranslational and transcriptional homeostatic regulatory mechanisms function together to maintain zinc at an optimal level under conditions of either zinc limitation or zinc excess. For example, in S. cerevisiae, expression of the high-affinity zinc uptake gene ZRT1 increases in response to zinc limitation, whereas under zinc-replete conditions, Zrt1 undergoes zinc-induced endocytosis and is degraded in the vacuole (39). However, unlike iron and copper, zinc-responsive transcription factors are found in fungi, mammals, fish, and possibly plants, suggesting that the transcriptional control of genes involved in zinc homeostasis is of universal importance (31, 58, 110, 151, 168). To date, two factors that control gene expression in response to zinc have been characterized in detail. These are Zap1 from S. cerevisiae, which activates gene expression in response to zinc deficiency, and mammalian MTF-1, which is activated by zinc (151, 168).

Under zinc-limiting conditions, Zap1 increases the expression of three zinc uptake systems encoded by the ZRT1, ZRT2, and FET4 genes (Fig. 1A) (149, 166, 167). Zap1 also stimulates the release of zinc from the vacuolar zinc store by activating the expression of the ZRT3 vacuolar efflux system (95). A fifth target of Zap1 is ZRC1, a gene that encodes a vacuolar zinc influx system (96). Although it seems counterintuitive that Zap1 up-regulates the expression of a gene associated with lowering cytoplasmic zinc levels, recent studies have revealed that the increased expression of ZRC1 in response to zinc limitation is a proactive mechanism to protect zinc-limited cells from possible exposure to high zinc levels (97). In addition to the five zinc transporter genes, Zap1 regulates the expression of 42 other genes, some of which may have additional roles in zinc homeostasis (Table 1) (94).

In mammals, MTF-1 plays a central role in protecting cells against zinc toxicity. This is partly achieved by increasing the expression of MT-1 and MT-2, two genes that encode zinc-binding metallothioneins (66). MTF-1 also lowers cytoplasmic zinc levels by regulating the expression of a zinc efflux system encoded by the ZnT-1 gene (87). A further putative target gene of MTF-1 is hZTL1, a gene that encodes a zinc uptake transporter that is localized to the enterocyte apical membrane (27). While increased hZTL1 expression may assist efficient uptake of zinc from a zinc-rich diet, this apparent regulation counteracts other homeostatic mechanisms. Perhaps this unusual regulation ensures that zinc is effectively absorbed from the intestine while other transcriptional and posttranslational homeostatic mechanisms maintain cellular zinc at an optimal level under these conditions.

In addition to regulating genes involved in zinc homeostasis, MTF-1 regulates the expression of a number of other genes (Table 1) (92). In mice, MTF-1 is an essential gene, with knockout mice dying in utero at approximately day 14 of gestation due to degeneration of hepatocytes (59). Although the exact reason for the lethality of the MTF-1 knockout is currently unknown, a number of candidate target genes of MTF-1 that are essential for embryonic development (C/EBP{alpha} and {alpha}-fetoprotein) could provide clues to the lethality phenotype (92). Contrary to the lethality of MTF-1 in mice, an MTF-1 knockout in Drosophila melanogaster is viable (38). In Drosophila, however, copper is a more potent inducer of MTF-1 activity than zinc and MTF-1 plays a dual role in regulating genes involved in resistance to copper toxicity and in preventing copper deficiency (38, 163). Thus, MTF-1 can have species-specific cellular roles in addition to zinc homeostasis.

Zap1 and MTF-1 have a number of features that are common to transcriptional activators, which include transactivation domains and DNA-binding domains containing C2H2-type zinc finger motifs (Fig. 3A). Zap1 contains two acidic activation domains (168). The first activation domain is located at the N terminus in a region rich in cysteine and histidine residues, and the second activation domain maps to a region containing two C2H2-type zinc finger domains (11). A further five C-terminal zinc finger domains are all essential for Zap1 DNA-binding activity (10, 40, 165). MTF-1 encodes a 72.5-kDa protein that contains six C2H2-type zinc finger domains and three transactivation domains (Fig. 3A) (21, 122).

A critical feature in understanding zinc homeostasis is determining how these factors sense zinc. Multiple regulatory mechanisms contribute to the inactivation of Zap1 by zinc (Fig. 3B). At the transcriptional level, Zap1 binds to a zinc-responsive element located within its own promoter and autoregulates its own expression. Zap1 activity is also regulated by up to three posttranslational mechanisms (12, 165, 168). The most understood regulatory mechanism is the autonomous repression of activation domain 2 (AD2) by zinc. Both of the zinc finger domains that are located in AD2 (Znf1 and Znf2) are required for zinc-responsive repression. In vitro, zinc binds with a slightly lower affinity and, notably, with a much higher lability to the Znf1-Znf2 pair relative to a control pair of zinc finger domains (11). Moreover, in vivo, residues that form the packing interface between the two fingers are an essential component of zinc-responsive repression. Thus, as zinc increases, the zinc occupancy of Znf1 and Znf2 may result in an interfinger, protein-protein interaction that masks critical residues that are essential for transactivation domain function (11).

In the absence of AD2 regulation, both AD1 and the Zap1 DNA-binding domain are negatively regulated by zinc by independent mechanisms (12). Repression of AD1 most likely involves a zinc-dependent intramolecular interaction with the Zap1 DNA-binding domain that masks activation domain function. In support of this model, AD1 is rich in potential zinc-coordinating ligands and repression of AD1 by zinc requires the Zap1 DNA-binding domain. In addition, a less-zinc-responsive mutant allele of ZAP1 (ZAP1-1up) encodes a cysteine-to-serine mutation in a region that is immediately upstream from AD1 (Fig. 3A) (12, 168). Zap1 DNA-binding activity may also be regulated by zinc, since the Zap1 DNA-binding domain is able to confer zinc-responsiveness onto a heterologous activation domain (12). At present, the precise mechanism by which zinc inhibits Zap1 DNA-binding activity is unknown.

Unlike Zap1, which is primarily regulated by zinc, MTF-1 activity can be regulated by zinc, other divalent metal ions, and various stress conditions in vivo (4, 51, 91). In vitro, zinc stimulates transcriptional induction by MTF-1, whereas induction in response to cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein (164). Moreover, in Drosophila, MTF-1 is primarily regulated by copper, whereas in transfected mammalian cells, Drosophila MTF-1 responds to zinc like mammalian MTF-1 (163). Thus, other aspects of metal ion homeostasis, such as metal release from metallothionein, may influence the primary metal specificity of MTF-1.

MTF-1 is regulated at multiple levels by zinc (Fig. 3C) (4, 51, 91). The first level of regulation of MTF-1 involves its cellular localization. Under noninducing conditions, MTF-1 predominantly resides in the cytoplasm. Upon addition of zinc or cadmium, MTF-1 rapidly translocates to the nucleus (128, 133). Mutation of the MTF-1 NES results in the retention of MTF-1 in the nucleus in a form that is able to bind to a metal response element (MRE) in response to zinc but is unable to activate transcription. Thus, a second level of regulation involving the MTF-1 cellular position could potentially be nucleocytoplasmic shuttling, i.e., MTF-1 might undergo a constant cycle of activation, nuclear import, deactivation, nuclear export, and then reactivation (128). The third level of regulation is the control of DNA-binding activity (13, 30, 66, 82). MTF-1 DNA-binding activity increases upon the addition of zinc. Consequently, one model is that one or more of the zinc finger domains bind zinc with a low affinity or low stability; thus, full DNA-binding activity is only achieved upon full metallation of the regulatory zinc finger(s) (66, 151).

Recent in vitro studies have clearly demonstrated that there is conformational heterogeneity in the MTF-1 DNA-binding zinc fingers such that the zinc bound to Znf5, Znf6, and to a lesser extent, Znf1 is less stable than the zinc bound to the remaining finger domains (Znf2, Znf3, and Znf4) (25, 26, 52). While Znf2, Znf3, and Znf4 form the core DNA-binding domain in vitro, deletion of Znf1 or Znf5 and Znf6 leads to attenuation of zinc-induced MTF-1 DNA binding at the endogenous MT-1 promoter in vivo (75). Thus, Znf1, Znf5, and Znf6 are required for maximal binding under zinc-replete conditions (75). Similar effects are not seen in mutants containing deletions of Znf5 or Znf6, whether examined in vitro or with an MRE-reporter construct, suggesting that binding zinc in these regulatory fingers may stabilize an MTF-1-chromatin complex (13, 75, 82).

MTF-1 activity is also controlled by phosphorylation. MTF-1 is phosphorylated in both an uninduced and an induced state. However, the level of phosphorylation is stimulated two- to fourfold by the addition of zinc. Kinase inhibitor studies have revealed that this phosphorylation is an essential component of zinc-responsive MTF-1 activation. Since kinase inhibitors have little effect on the nuclear import or DNA-binding activity of MTF-1, signal transduction pathways may use phosphorylation or dephosphorylation to control activation domain function (88, 127). Finally, under specific conditions, other factors can influence MTF-1 activity. The precise activity of MTF-1 may also be dependent on the MRE sequence, the chromatin architecture of the target loci, cell type, developmental stage of growth, and growth conditions (1, 5, 50, 104).

Both Zap1 and MTF-1 use regulatory zinc finger domains to sense zinc; however, it is currently unknown what properties of a zinc finger make it regulatory. The regulatory zinc fingers all match the consensus zinc finger sequence (Phe/Tyr-X-CysX2-4-Cys-X3-[Phe]-X5-Leu-X2-His-X2-3-His), with the exception of the conserved central phenylalanine. In Znf1 and Znf2 from Zap1, this residue is a cysteine and glycine, respectively. Attempts to convert the Zap1, Znf1, and Znf2 fingers back to the consensus sequence (by converting the cysteine and glycine residues at the finger tip to phenylalanine) have no effect on AD2 activity, suggesting that the high lability of the zinc bound to these fingers is not simply caused by these substitutions (11). Importantly, these zinc fingers function together as a pair, suggesting that other residues that stabilize pair formation may have important regulatory functions. In MTF-1, Znf5 fluctuates between the canonical ßß{alpha} structure and another structure or structures upon addition of excess zinc (52). Znf5 contains five additional potential zinc-coordinating ligands that are located in the Cys-X4-Cys loop and the {alpha}-helix. The high conservation of these residues and the properties of Znf5 have led to the speculation that these residues may bind an additional zinc ion that destabilizes Znf5 (52). Thus, a precise knowledge of what makes these particular fingers bind zinc with a higher lability than other fingers will help us to understand their regulatory function.

Another unanswered question is what zinc pools do MTF-1 and Zap1 sense. In Escherichia coli, the zinc sensors Zur and ZntR respond to femtomolar levels of zinc (less than 1 atom of zinc per cell) (106). These data suggest that the majority of cellular zinc is bound in a yet-unidentified bioavailable zinc pool that could consist of proteins, small molecules, or other macromolecules that either strongly or weakly chelate zinc. Because of the predicted low levels of free zinc, it has been proposed that zinc-trafficking factors may be required to deliver zinc to proteins (106). Although the precise level of free zinc in eukaryotic cells is unknown, it is interesting that both MTF-1 and Zap1 sense zinc in the nanomolar to subnanomolar range (11, 52). If free intracellular zinc levels are maintained at much less than nanomolar concentrations in eukaryotic cells, then the eukaryotic zinc sensors must be detecting fluctuations in a bioavailable zinc pool. Studies with eukaryotic sensors may therefore help us to answer questions such as what molecule(s) or protein(s) potentially delivers zinc to Zap1 and MTF-1. These studies also raise many other interesting questions, such as whether all zinc-sensing factors rely on regulatory zinc fingers or whether other types of zinc domains, such as the GATA or LIM domain, can be used.

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