Cellular responses to zinc involving the transcription factor ZNF658 and its target genes

Abstract

Zinc is an essential trace element that plays a crucial role in catalytic, structural and regulatory functions of many proteins including enzymes and transcription factors; thus maintenance of zinc balance is critical for normal cellular function. Cellular mechanisms that maintain zinc balance include the regulation of genes coding for proteins that play vital roles in zinc homeostasis. These proteins include zinc transporters belonging to the ZIP (SLC39A) and ZnT (SLC30A) families as well as the zinc-binding metallothionein proteins. In contrast to bacterial and yeast systems, a transcription factor responsible for mediating transcriptional repression of a suite of genes in response to elevated zinc levels in mammals has hitherto not been identified. Using Caco-2 cells as a model of human intestinal epithelial cells and detection of protein binding by electrophoretic mobility shift analysis, we show that zinc finger protein ZNF658 binds specifically to the zinc transcriptional regulatory element (ZTRE), previously demonstrated to mediate this response in a panel of three genes: SLC30A5 (ZnT5 zinc transporter), SLC30A10 (ZnT10 zinc transporter) and CBWD (whose prokaryotic homologs are emerging players in metal biology). We also demonstrate that siRNA-driven reduction of ZNF658 attenuated or abrogated transcriptional repression in response to elevated zinc levels of these same genes by measuring transcript abundance using RT-qPCR and using promoter-reporter gene constructs. In addition, the region of ZNF658 responsible for binding to the ZTRE was identified (the C-terminal zinc finger domain) and the requirement for both sides of the palindromic ZTRE sequence for function was demonstrated. This study therefore identifies the first metazoan transcription factor that plays a pivotal role in the orchestrated cellular response to increased zinc levels to restore cellular zinc balance necessary to achieve a broad spectrum of zinc-dependent functions and begins to probe its molecular action. We also report an important role for the mammalian CBWD gene product in protection of cells from either depleted or excess zinc by virtue of the fact that overexpression of recombinant CBWD protein altered cellular tolerance to both elevated and depleted levels of zinc consistent with a homeostatic function. In addition, we present preliminary evidence that changes in the expression of ZNF658 and its target genes, in particular SLC30A10 (ZnT10), may be related to cell senescence, suggesting that changes in zinc homeostasis are components of this process. Overall, the work presented contributes to understanding zinc regulated gene expression and cellular zinc homeostasis.