Amanda J. Bird

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

Iron, copper, and zinc are all essential nutrients. The electron transfer properties of iron and copper are fundamental to processes such as respiration and photosynthesis. Zinc forms the catalytic center in numerous enzymes and has an important structural role in a wide range of proteins. However, all these metals can be toxic if their levels and distribution are not carefully regulated, as their inappropriate binding may compromise cellular function. The uncontrolled redox activity of iron and copper can also lead to the generation of damaging oxygen radicals. Therefore, organisms maintain cytoplasmic metal concentrations at a nontoxic level that is sufficient for growth. A variety of homeostatic mechanisms have been identified, which include the control of translation and RNA stability by iron-regulatory proteins and the metal-dependent trafficking or degradation of metal transporters (39, 109, 138). This review focuses on the role that metal-responsive transcription factors have in regulating trace metal metabolism. These factors are able to sense changes in metal concentrations and coordinate the expression of genes that are involved in the acquisition, distribution, sequestration, and use of metals. Consequently, the ability to mediate metal-responsive gene expression is an important aspect of metal homeostasis in those organisms that contain these factors.

Repression of ADH1 and ADH3 during zinc deficiency by Zap1-induced intergenic RNA transcripts

The transcriptional activator Zap1 induces target gene expression in response to zinc deficiency. We demonstrate that during zinc starvation, Zap1 is required for the repression of ADH1 expression. ADH1 encodes the major zinc‐dependent alcohol dehydrogenase that is utilized during fermentation. During zinc starvation, Zap1 binds upstream of the activator Rap1 and induces an intergenic RNA transcript, ZRR1. ZRR1 expression leads to the transient displacement of Rap1 from the ADH1 promoter resulting in ADH1 repression. Using a microarray‐based approach, we screened for additional genes repressed by Zap1 intergenic transcripts. We found that ADH3, the major mitochondrial alcohol dehydrogenase, is regulated in a manner similar to ADH1. Thus, during zinc deficiency, Zap1 mediates the repression of two of the most abundant zinc‐requiring enzymes.

Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function

The yeast Zap1 transcription factor controls the expression of genes involved in zinc accumulation and storage. Zap1 is active in zinc‐limited cells and repressed in replete cells. Zap1 has two activation domains, AD1 and AD2, which are both regulated by zinc. AD2 function was mapped to a region containing two Cys2His2 zinc fingers, ZF1 and ZF2, that are not involved in DNA binding. More detailed mapping placed AD2 almost precisely within the endpoints of ZF2, suggesting a role for these fingers in regulating activation domain function. Consistent with this hypothesis, ZF1 and ZF2 bound zinc in vitro but less stably than did zinc fingers involved in DNA binding. Furthermore, mutations predicted to disrupt zinc binding to ZF1 and/or ZF2 rendered AD2 constitutively active. Our results also indicate that the repressed form of AD2 requires an intramolecular interaction between ZF1 and ZF2. These studies suggest that these zinc fingers play an unprecedented role as zinc sensors to control activation domain function.

Evidence for a Pro-oxidant Intermediate in the Assembly of Cytochrome Oxidase

The hydrogen peroxide sensitivity of cells lacking two proteins, Sco1 and Cox11, important in the assembly of cytochrome c oxidase (CcO), is shown to arise from the transient accumulation of a pro-oxidant heme A-Cox1 stalled intermediate. The peroxide sensitivity of these cells is abrogated by a reduction in either Cox1 expression or heme A formation but exacerbated by either enhanced Cox1 expression or heme A production arising from overexpression of COX15. Sco1 and Cox11 are implicated in the formation of the CuA and CuB sites of CcO, respectively. The respective wild-type genes suppress the peroxide sensitivities of sco1Δ and cox11Δ cells, but no cross-complementation is seen with noncognate genes. Copper-binding mutant alleles of Sco1 and Cox11 that are nonfunctional in promoting the assembly of CcO are functional in suppressing the peroxide sensitivity of their respective null mutants. Likewise, human Sco1 that is nonfunctional in yeast CcO assembly is able to suppress the peroxide sensitivity of yeast sco1Δ cells. Thus, a disconnect exists between the respiratory capacity of cells and hydrogen peroxide sensitivity. Hydrogen peroxide sensitivity of sco1Δ and cox11Δ cells is abrogated by overexpression of a novel mitochondrial ATPase Afg1 that promotes the degradation of CcO mitochondrially encoded subunits. Studies on the hydrogen peroxide sensitivity in CcO assembly mutants reveal new aspects of the CcO assembly process.

A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc‐limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post‐translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA‐binding activity is conferred by five C‐terminal C2H2 zinc fingers and each finger is required for high‐affinity DNA binding. The zinc‐responsive domain of Zap1 also maps to the C‐terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA‐binding domain–Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.

Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae

BackgroundThe Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes.ResultsWith alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses.ConclusionWe have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.

Regulation of the yeast TSA1 peroxiredoxin by ZAP1 is an adaptive response to the oxidative stress of zinc deficiency

Zinc deficiency is a potential risk factor for disease in humans because it leads to increased oxidative stress and DNA damage. We show here that the yeast Saccharomyces cerevisiae also experiences oxidative stress when zinc-deficient, and we have identified one mechanism yeast cells use to defend themselves against this stress. The Zap1p transcription factor is a central player in the response of yeast to zinc deficiency. To identify genes important for growth in low zinc, DNA microarrays were used to identify genes directly regulated by Zap1p. We found that the TSA1 gene is one such Zap1p target whose expression is increased under zinc deficiency. TSA1 encodes a cytosolic thioredoxin-dependent peroxidase responsible for degrading hydrogen peroxide and organic hydroperoxides. Consistent with its regulation by Zap1p, we showed that tsa1Δ mutants have a growth defect in low zinc that can be suppressed by zinc but not by other metals. Anaerobic conditions also suppressed the tsa1Δ low zinc growth defect indicating that oxidative stress is the likely cause of the poor growth. Consistent with this hypothesis, we demonstrated that zinc deficiency causes increased reactive oxygen species in wild type cells and that this increase is further exacerbated in tsa1Δ mutants. The role of this regulation by Zap1p in limiting oxidative stress in low zinc was confirmed when the Zap1p-binding site was specifically mutated in the chromosomal TSA1 promoter. Thus, we conclude that TSA1 induction by Zap1p is an adaptive response to deal with the increased oxidative stress caused by zinc deficiency.

The Zap1 transcriptional activator also acts as a repressor by binding downstream of the TATA box in ZRT2

The zinc‐responsive transcriptional activator Zap1 regulates the expression of both high‐ and low‐affinity zinc uptake permeases encoded by the ZRT1 and ZRT2 genes. Zap1 mediates this response by binding to zinc‐responsive elements (ZREs) located within the promoter regions of each gene. ZRT2 has a remarkably different expression profile in response to zinc compared to ZRT1. While ZRT1 is maximally induced during zinc limitation, ZRT2 is repressed in low zinc but remains induced upon zinc supplementation. In this study, we determined the mechanism underlying this paradoxical Zap1‐dependent regulation of ZRT2. We demonstrate that a nonconsensus ZRE (ZRT2 ZRE3), which overlaps with one of the ZRT2 transcriptional start sites, is essential for repression of ZRT2 in low zinc and that Zap1 binds to ZRT2 ZRE3 with a low affinity. The low‐affinity ZRE is also essential for the ZRT2 expression profile. These results indicate that the unusual pattern of ZRT2 regulation among Zap1 target genes involves the antagonistic effect of Zap1 binding to a low‐affinity ZRE repressor site and high‐affinity ZREs required for activation.

Independent Metalloregulation of Ace1 and Mac1 in Saccharomyces cerevisiae

ABSTRACT Ace1 and Mac1 undergo reciprocal copper metalloregulation in yeast cells. Mac1 is functional as a transcriptional activator in copper-deficient cells, whereas Ace1 is a transcriptional activator in copper-replete cells. Cells undergoing a transition from copper-deficient to copper-sufficient conditions through a switch in the growth medium show a rapid inactivation of Mac1 and a corresponding rise in Ace1 activation. Cells analyzed after the transition show a massive accumulation of cellular copper. Under these copper shock conditions we show, using two epitope-tagged variants of Mac1, that copper-mediated inhibition of Mac function is independent of induced protein turnover. The transcription activity of Mac1 is rapidly inhibited in the copper-replete cells, whereas chromatin immunoprecipitation studies showed only partial copper-induced loss of DNA binding. Thus, the initial event in copper inhibition of Mac1 function is likely copper inhibition of the transactivation activity. Copper inhibition of Mac1 in transition experiments is largely unaffected in cells overexpressing copper-binding proteins within the nucleus. Likewise, high expression of a copper-binding, non-DNA-binding Mac1 mutant is without effect on the copper activation of Ace1. Thus, metalloregulation of Ace1 and Mac1 occurs independently.

Mapping the DNA Binding Domain of the Zap1 Zinc-responsive Transcriptional Activator

The Zap1 transcriptional activator ofSaccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Δ mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.