Abstract
Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a specific sequence, the zinc transcriptional regulatory element (ZTRE), in the SLC30A5 (zinc transporter ZnT5) promoter and showed that substitution of the ZTRE abrogated the repression of a reporter gene in response to zinc. We identified the ZTRE in other genes, including (through an unbiased search) the CBWD genes and (through targeted analysis) in multiple members of the SLC30 family, including SLC30A10, which is repressed by zinc. The function of the CBWD genes is currently unknown, but roles for homologs in metal homeostasis are being uncovered in bacteria. We demonstrated that CBWD genes are repressed by zinc and that substitution of the ZTRE in SLC30A10 and CBWD promoter-reporter constructs abrogates this response. Other metals did not affect expression of the transcriptional regulator, binding to the ZTRE or promoter-driven reporter gene expression. These findings provide the basis for elucidating how regulation of a network of genes through this novel mechanism contributes to zinc homeostasis and how the cell orchestrates this response.
Highlights
Coordinated gene transcription is essential to zinc homeostasis
Electrophoretic Mobility Shift Assay (EMSA)—An infrared dye (IRD)-labeled probe corresponding to the SLC30A5 promoter (Ϫ156 to ϩ46 relative to the transcription start site) was generated from the plasmid pBlueSLC30A5prom in a PCR (HotStart Taq Mastermix, Qiagen) using thermal cycling parameters and primers (0.5 M each) with an attached 5Ј-IRD molecule (Eurofins MWG Operon) as specified in supplemental Table S1
We have established that zinc-mediated transcriptional repression of the human SLC30A5 gene involves the zinc-dependent binding of a zinc-regulated protein factor to a palindromic sequence, the zinc transcriptional regulatory element (ZTRE), at positions Ϫ91 to Ϫ76 bp upstream of the end of the 5Ј-UTR, defining a novel mechanism for zinc-mediated transcriptional regulation
Summary
Coordinated gene transcription is essential to zinc homeostasis. Results: The human ZTRE binds a zinc-responsive transcriptional regulator and is in genes with roles in zinc-related functions. Mutation of the consensus MRE sequence in a zinc-repressed promoter-reporter construct based on the upstream region of the gene SLC30A5, coding for the human zinc transporter ZnT5, failed to abolish zinc-induced transcriptional repression in transfected human intestinal Caco-2 cells [21], indicating a mechanism of zinc-responsive transcriptional regulation independent of MTF1. KLF4 (Krüppel-like factor 4) appears to have a role in regulation of the zinc transporter Zip (gene Slc39a4) in mouse intestine, as indicated by observations including zinc-sensitive binding of KLF4 to a region of the promoter in vitro, the requirement for a KLF4 binding sequence in a Zip promoter-reporter construct for effects of zinc on reporter gene expression, and curtailment of Zip induction by zinc limitation in a mouse intestinal cell line by KLF4 knockdown [22]. We present the first direct evidence for a transcriptional regulatory process independent of MTF1 that operates in mammalian cells to repress transcription of multiple genes with diverse functions in response to increased zinc availability
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