Zinc is the second most abundant transition metal in humans and is essential for life. Zinc is required for the activity of more than 300 enzymes spanning the six major classes of enzymes. Also, the coordination of zinc ions confers proper structural conformation to numerous proteins. Given the importance of zinc to cellular metabolism, zinc deficiency is harmful to health. Zinc, in excess, is also toxic to cells. Therefore, all organisms have evolved zinc homeostasis mechanisms to maintain an optimum range of intracellular zinc levels. Zinc-responsive transcriptional regulation is one of the most common means to achieve zinc homeostasis. Transcriptional factors can regulate the expression of zinc transporters, zinc-sequestering proteins, and abundant zinc-binding proteins according to cellular zinc status. Most of our knowledge about the zinc-responsive transcriptional regulation in eukaryotes is derived from Zap1 from Saccharomyces cerevisiae and MTF-1 from vertebrates, which activate gene expression in response to zinc deficiency and excess zinc, respectively. However, there are several questions remaining to be answered, including the regulation of mammalian zinc-regulated genes independent of MTF-1 and the properties that enable proteins to sense intracellular zinc levels. The fission yeast Schizosaccharomyces pombe lacks a Zap1 homolog, and yet regulates gene expression in response to zinc. It was later discovered that Loz1 was required for this zinc-responsive transcriptional regulation of gene expression.
In this study, I have investigated the role of Loz1 in the zinc-responsive regulatory pathway. The results from this study show that Loz1 is a transcriptional factor that represses the expression of target genes by binding to their promoters under zinc-replete conditions. For the Loz1-mediated regulation in vivo, a Loz1 Response Element (LRE) with the sequence 5’-CGNMRATCNTY-3’ is necessary and sufficient. By combining information about the Loz1 binding loci to the transcriptomic changes that occur in a Loz1-dependent manner, I also determined the Loz1 regulon under zinc-replete growth conditions. By expressing two predicted Loz1 orthologs in S. pombe cells lacking Loz1, I found that these proteins, having the conserved C2H2-type zinc fingers and divergent accessory domains, retained the ability to regulate the expression of Loz1 target genes in a zinc-dependent manner.
In the second part of this study, I elucidated the role of the Ecl family proteins in the regulation of two highly regulated Loz1 target genes, zrt1 and SPBC1348.06c. I found that the Ecl family-mediated activation of these genes is independent of the zinc-sensing regulatory pathway that contains Loz1.
In the final chapter of this work, I determined the subcellular localization of Zhf1, which is a zinc transporter that is required for growth under excess zinc. Fluorescence microscopy using GFP-tagged Zhf1 showed a vacuolar membrane localization, which suggests that excess zinc is stored in vacuoles where the bulk of cellular phosphorus is also stored.
The data presented within this dissertation confirm Loz1 as the first zinc-responsive transcriptional repressor identified in eukaryotes. Since the Loz1 DNA binding in vivo is zinc-dependent and this DNA binding requires the two conserved C-terminal C2H2-type zinc fingers, our results strongly suggest the zinc-responsive regulatory role to be due to the ability of the Loz1 zinc fingers to `sense’ intracellular zinc status.