Only a small number of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater number of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.

Changes in DNA methylation across the life course may contribute to the ageing process. We hypothesised that some effects of dietary restriction to extend lifespan and/or mitigate against features of ageing result from changes in DNA methylation, so we determined if genes that respond to dietary restriction also show age-related changes in DNA methylation. In support of our hypothesis, the intersection of lists of genes compiled from published sources that (1) were differentially expressed in response to dietary restriction and (2) showed altered methylation with increased age was greater than expected. We also hypothesised that some effects of Sirt1, which may play a pivotal role in beneficial effects of dietary restriction, are mediated through DNA methylation. We thus measured effects of Sirt1 overexpression and knockdown in a human cell line on DNA methylation and expression of a panel of eight genes that respond to dietary restriction and show altered methylation with age. Six genes were affected at the level of DNA methylation, and for six expressions were affected. In further support of our hypothesis, we observed by DNA microarray analysis that genes showing differential expression in response to Sirt1 knockdown were over-represented in the complied list of genes that respond to dietary restriction. The findings reveal that

An age-associated decrease in zinc status has been observed in several studies but the underlying causes remain uncertain. A decrease in zinc absorption due to changes in expression of zinc transporters may be a contributory factor. Zinc dyshomeostasis, including altered zinc transporter expression, has been associated also with several diseases of ageing, including Alzheimer's disease, prostate cancer, type-2 diabetes and macular degeneration. Mechanisms responsible for age-and disease-associated changes in zinc transporter expression are still to be uncovered.The zinc transcriptional regulatory element (ZTRE) represses gene expression at high levels of zinc availability through the binding of a transcriptional regulatory factor, and has been shown to be functional in the SLC30A5 and SL30A10 human zinc transporter genes (1) . After identifying the ZTRE we noted that a CpG site in the SLC30A5 promoter at which we previously observed an age-related increase in methylation in human intestine (2) is within the ZTRE. Methylation of this CpG site reduced binding of the regulatory protein, and the inhibitory effect was augmented when three additional CpG sites within 50 base pairs of the ZTRE were also methylated (1) .We propose that age-related epigenetic modifications, including DNA methylation, contribute to changes in the expression of zinc transporters to affect zinc homeostasis. We propose specifically that age-associated changes in DNA methylation at CpG sites within or in close proximity to ZTREs in the promoter regions of other zinc transporter genes affect the binding of transcription factors, as observed for SLC30A5, thus contributing to impaired zinc homeostasis in the ageing gut and to zinc-related disease processes in other tissues.Bioinformatic analysis using Fuzznuc software identified ZTREs within 1 kb upstream of the transcription start site of the human SLC30A1, SLC30A3, SLC30A4, SLC30A7 and the mouse Slc30a4 and Slc30a5 zinc transporter genes. Using pyrosequencing, we measured DNA methylation around the ZTRE in the Slc30a5 gene in samples of young (4-8 mo; n = 6-7) and old (32-36 mo; n = 4) mouse intestine. In contrast to our findings in human samples, there was no difference in methylation of the CpG site within the Slc30a5 ZTRE or of 7 CpG sites within 28 base pairs of this site between the two groups (Kruskal-Wallis P = 0.571). Future work will include repeating these measurements on a larger number of samples and also extending the analysis to the mouse Slc30a4 gene and to human zinc transporter genes additional to SLC30A5.These preliminary findings, in light of the established effect on protein binding to the ZTRE, indicate that age-related changes in DNA methylation at specific CpG sites within or close to ZTREs in genes with roles in zinc homeostasis may affect zinc homeostasis in older age. Should analysis of larger numbers of samples reveal robust associations between age and methylation at CpG sites within and close to ZTREs it will be essential to establish if these changes affect gene e...