Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1

Cell and gene therapy for friedreich ataxia: Progress to date

Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since individuals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease

Zinc regulation of Zap1 ZRE binding maps to the DNA binding domain.

<p><b>A</b>) Diagram of wild-type Zap1 and Zap1^Δ17-700. The latter allele retains only a small part of the N-terminus of the protein and the intact DNA binding domain. The <i>hatched</i> boxes indicate the activation domains and the <i>black</i> boxes numbered 1–7 denote the zinc fingers. <b>B</b>) ZHY6 <i>zap1Δ</i> cells transformed with pPGK-ZRT1, and either pZap1^WT, pZap1^Δ17-700, or the pYef2 empty vector were grown to exponential phase in LZM supplemented with 3 µM or 1000 µM ZnCl₂. pPGK-ZRT1 expresses the high affinity <i>ZRT1</i> zinc transporter from the <i>PGK1</i> promoter allowing a cell with no functional Zap1 to grow well in low zinc. Total protein extracts were prepared and subjected to immunoblot analysis using antibodies against Zap1 (myc) and Pgk1. <b>C</b>) The same cells as described in panel B were grown to exponential phase and chromatin immunoprecipitation analysis was performed using primers flanking the ZRE in <i>ZRT1</i>. Primers specific for the promoter region of <i>CMD1</i> were used as a negative control. The shown inputs are 1000-fold dilutions of whole cell extracts and 10-fold serial dilutions of representative samples are included to confirm the quantitative nature of the assay.</p

Possible zinc regulation of the Zap1 DNA binding domain <i>in vivo</i>.

<p><b>A</b>) Wild-type (DY1457) or <i>zap1Δ</i> (ZHY6) cells transformed with either pZap1^WT, pZap1^TC, or the pYef2 vector and the pGI-1 <i>ZRT1-lacZ</i> reporter were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂ prior to analysis of β-galactosidase activity. Zap1^TC has mutations in the zinc-responsive domains of Zap1's two activation domains rendering those domains constitutive. <i>ZRT1</i> encodes a plasma membrane zinc uptake transporter and is a Zap1 target gene. Values plotted are the means of three replicate cultures and the error bars indicate ±1 S.D. <b>B</b>) Total RNA was extracted from the cells described in panel A grown in LZM supplemented with 3 µM or 1000 µM ZnCl₂ and S1 nuclease assays were performed to determine mRNA levels of <i>ZRT1</i> and <i>ZPS1</i>, another Zap1 target gene that encodes a secreted protein of unknown function. <i>CMD1</i> was used as a loading control. <b>C</b>) Graphical representation of results obtained in panel B.</p

The nuclear localization of Zap1 is not affected by zinc.

<p>Protease-deficient BJ2168 cells transformed with pZap1^WT were grown to exponential phase in LZM supplemented with 3 µM or 1000 µM ZnCl₂. BJ2168 cells lacking pZap1^WT were also grown in LZM supplemented with 1000 µM ZnCl₂. Total cell homogenates were separated into cytosolic and nuclear fractions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#s4" target="_blank">Materials and Methods</a>. Equal amounts of protein from each sample (10 µg protein/lane) were assayed by immunoblotting using antibodies against Zap1 (myc), Pgk1, Dpm1, and Pho2.</p

Overexpressing Zap1 disrupts gene regulation and zinc homeostasis.

<p><b>A</b>) ZHY6 <i>zap1Δ</i> cells transformed with pGEV, the pGI-1 <i>ZRT1-lacZ</i> reporter and either pZap1^WT or pYef2 vector were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂. To induce high Zap1 expression, cells were treated with 1 µM β-estradiol (ox). Cells were then harvested and β-galactosidase assays were performed. <b>B</b>) Wild-type DY1457 cells transformed with pGEV, the pGI-1 <i>ZRT1-lacZ</i> reporter, and either pYef2 or pZap1^mZnf4 were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂. To induce high Zap1^mZnf4 levels, cells were treated with 1 µM β-estradiol (ox). Cells were then harvested and β-galactosidase assays were performed. The values shown in panels A and B are the means of three independent cultures, and the error bars equal ±1 S.D. <b>C</b>) Protein extracts were generated from the samples described in panels A and B and subjected to immunoblot analysis using antibodies against Zap1 (myc) and Pgk1. <b>D</b>) Wild-type DY1457 cells and ZHY6 <i>zap1Δ</i> cells transformed with pGEV and either the pYef2 vector or pZap1^WT were inoculated at an A₆₀₀ of 0.02 in LZM supplemented with the indicated concentration of ZnCl₂ plus tracer amounts of ⁶⁵ZnCl₂. To induce high levels of Zap1^WT protein expression, cultures were treated with 1 µM β-estradiol (Zap1^WTox). Cultures were grown to an A₆₀₀ of ∼0.75, after which zinc accumulation was measured. Shown are the means of three independent cultures and the error bars indicate ±1 S.D. The <i>asterisks</i> indicate a significant difference (<i>p</i><0.05) of <i>zap1Δ</i> cells expressing Zap1^WTox relative to Zap1^WT controls as determined by 2-sided Student <i>t</i>-test.</p

Confirmation of regulated Zap1 ZRE occupancy by chromatin immunoprecipitation.

<p>ZHY6 <i>zap1Δ</i> cells transformed with pZap1^WT were grown to exponential phase in LZM+3 µM or 1000 µM ZnCl₂. Wild-type (DY1457) cells transformed with pYef2 vector were used as a negative control. Cells were then harvested and chromatin immunoprecipitation was performed using primers flanking the ZREs in <i>ZRT1</i> and <i>ZPS1</i>. Primers specific for the promoter region of <i>CMD1</i> were used as a negative control. Shown inputs are 1000-fold dilutions of whole cell extracts, and 10-fold serial dilutions of representative samples were also PCR amplified to confirm the quantitative nature of the assay.</p

ZRE occupancy by Zap1 is altered by zinc status <i>in vivo</i>.

<p><b>A</b>) Wild-type (DY1457) cells were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂ and analyzed by <i>in vivo</i> DMS footprinting on the <i>ZRT1</i> promoter. A sample from a ZHY6 <i>zap1Δ</i> mutant grown in LZM+3 µM ZnCl₂ is shown for comparison. The position of the ZRE was determined using a DNA sequencing ladder (not shown) and is indicated by the <i>box</i>. The sequence of the ZRE is shown with the distance of those bases from the ATG start codon; the purines that are sensitive to DMS methylation are <i>italicized</i>. The ZRE bands used to quantify protection are marked by <i>asterisks</i> and <i>boxed</i>. <b>B</b>) The experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#pone-0022535-g002" target="_blank">Figure 2A</a> was repeated a total of six times and quantified as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#s4" target="_blank">Materials and Methods</a>. The mean percent protection levels are shown and the error bars indicate 1 S.D. <b>C</b>) Immunoblot analysis of Zap1 and Pgk1 levels in the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#pone-0022535-g002" target="_blank">Figure 2A</a>. Total protein extracts were prepared from aliquots of cells harvested prior to DMS treatment.</p

Zap1 DNA binding is controlled <i>in vivo</i> without autoregulation.

<p><b>A</b>) ZHY6 <i>zap1Δ</i> mutant cells transformed with either pZap1^WT or the vector pYef2 were grown to exponential phase in LZM+3 µM and LZM+1000 µM ZnCl₂. LZM contains 2% glucose as carbon source so expression of Zap1 from the <i>GAL1</i> promoter is at low levels similar to endogenous Zap1. Total protein extracts were prepared and subjected to immunoblot analysis using antibodies against Zap1 (myc) and Pgk1. <b>B</b>) ZHY6 <i>zap1Δ</i> cells transformed with the vector (pYef2) or pZap1^WT were grown to exponential phase in LZM+3 or 1000 µM zinc. Cells were than analyzed by <i>in vivo</i> DMS footprinting. The <i>box</i> indicates the position of the ZRE and the bands used for quantification of protection. <b>C</b>) The experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#pone-0022535-g003" target="_blank">Figure 3B</a> was repeated a total of six times and quantified as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#s4" target="_blank">Materials and Methods</a>. The mean percent protection levels are shown and the error bars indicate 1 S.D. Data for wild-type cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022535#pone-0022535-g002" target="_blank">Figure 2B</a> are shown again here for comparison.</p