Characterisation of a novel transcriptional mechanism for the regulation of mammalian gene expression by zinc

This research project is aimed towards increasing our understanding of fundamental molecular mechanisms through which gene expression is regulated in response to zinc. It was estimated recently that up to 10% of all human genes may code for proteins that require zinc to stabilise their structure or for their enzymatic function, indicating the importance of this element in healthy cell function. Zinc can not be produced in the body so it is an essential component of the diet, required in trace quantities. In the UK, subclinical zinc deficiency may contribute to health problems associated with reduced immune function and to poor pregnancy outcome. Worldwide, clinical zinc deficiency, observed in populations subsisting on zinc-poor, cereal-based diets, is a major health problem. The World Health Organisation identifies zinc, along with iron and vitamin A, as a micronutrient for which the population worldwide suffers acute deficit. It is important that conditions of zinc deficiency and zinc excess are avoided at both the whole body level and at the level of the individual cell, otherwise normal biological function can not be maintained. Homeostatic mechanisms, therefore, operate at the molecular level to regulate the expression of key genes involved in zinc transport, binding and storage. Mechanisms through which the expression of a gene can be regulated include changing the rate at which the cell makes RNA copies of that gene (i.e. changing the rate of gene transcription). The mechanism through which the cell increases the transcription of genes that must be expressed at increased levels in the presence of excess zinc (to remove zinc from the cell or to increase the ability of the cell to buffer the excess zinc) is fairly well understood but, in spite of much interest, the mechanism through which excess zinc reduces the transcription of other genes, involved in zinc uptake into the cell or in the release of stored zinc, is unknown. This project will use a range of different molecular techniques to study the regulation by zinc of the gene coding for the human zinc transporter ZnT5. We have found that this gene is regulated by increased zinc availability through reduced transcription, providing an ideal model system on which to study this process. Having identified the molecular components of this gene regulatory process, we will identify other genes whose regulatory regions include the same elements and determine if these are regulated by zinc in the same manner, to establish the extent to which the mechanism we identify is applicable to gene regulation by zinc more globally. The outcomes of the research will make a major contribution to our understanding of the fundamental mechanisms underlying cellular zinc homeostasis and will be of wide interest to researchers in the field of gene regulation in general and the cell biology of zinc in particular. In the specific context of dietary zinc absorption and zinc excretion, the findings will inform more applied research aimed towards optimising zinc nutrition, which has important implications for health worldwide.

Zinc is an essential micronutrient over which homeostasis is maintained through zinc-regulated gene expression. Up to 10% of all human genes may code for proteins that bind zinc and sub-optimal zinc nutrition is important worldwide. Mechanisms of zinc-regulated gene expression, other than transcriptional up-regulation, are poorly characterised. The aim of this project is to characterise a novel transcriptional mechanism for the repression of mammalian gene expression by zinc and to evaluate the extent to which this has a general role. The human zinc transporter ZnT5 (SLC30A5 gene) provides a model system on which to study zinc-induced transcriptional repression, for which a mechanism has not yet been described in mammalian cells. The hypothesis is that (a) protein factor(s) bind(s) in a zinc-dependent manner to a consensus sequence element, included in the SLC30A5 (ZnT5) promoter, and is/are pivotal in the down-regulation in response to zinc of a raft of genes regulated in this manner in mammalian cells. Binding sites for protein factors responsible for zinc-induced transcriptional down-regulation of the SLC30A5 gene will be identified by electrophoretic mobility shift analysis, by mutation of indicated protein binding sites in promoter-reporter gene constructs and by the introduction of multiple copies of potential binding motifs into a zinc-refractory promoter-reporter gene construct. Identification of proteins binding to the sequence(s) defined will be by mass spectrometry and/or by screening a cDNA expression library. Bioinformatics-based genome analysis and chromatin immunoprecipitation will identify other genes regulated through this same mechanism, selected examples of which will be confirmed directly. The findings will provide fundamental knowledge about the mechanisms through which cells maintain zinc homeostasis through the regulation of gene expression and will inform studies directed towards optimising zinc nutrition.