Background Premature and IUGR infants are often found to be zinc deficient, and zinc deficiency (ZnD) has been shown to decrease growth and increase morbidity. The molecular mechanisms responsible for this remain unknown. However, in our lab, we have seen that juvenile rats fed a zinc deficient diet are characterized by altered hepatic DNA methylation and expression of genes central to glucose homeostasis. To limit confounders invariably present in an in vivo system, we have developed a cell culture model of ZnD to isolate the effects of ZnD upon the epigenetic regulation of gene expression.
Objective We hypothesized that, in hepatic cell culture, ZnD would alter DNA methylation, nuclear heterochromatin, and gene expression.
Design/Methods Zinc deficient cell culture media was created by chelation with a resin specific for divalent cations. The resultant media was replenished with Ca2+, Mg2+, Fe2+, and Cu2+ so that the only difference was Zn2+ concentration (1μM vs 7.5μM). Cells were grown for 7 days and harvested for analysis. Cytosine extension assay was done to determine DNA methylation status using BssHII and HpaII. BssHII is a methylation sensitive enzyme that cuts a site frequently found within promoter regions, while the site for the methylation sensitive enzyme HpaII is widely dispersed throughout the genome. MspI was used as a non-methylation sensitive control. Nuclear heterochromatin was assessed using electron microscopy. mRNA levels for PGC-1, PEPCK, and GLUT2 were quantified using real-time RT-PCR.
Results ZnD increased CpG methylation. Methylation at BssHII sites was increased by 255±70%*, while methylation at HpaII was increased by 212±21%*. There was no significant difference noted in the MspI digests. EM revealed an increased amount of heterochromatin in zinc deficient vs control cells. Correlated with CpG hypermethylation and increased heterochromatin, mRNA levels of PGC-1, and PEPCK were decreased in zinc deficient cells by 26.9±3%* and 46.3±4%*, respectively. There was no significant change in GLUT2 expression. *(n=3, p≤0.05)
Conclusions In hepatic cell culture, ZnD increases DNA methylation and nuclear heterochromatin, and decreases expression of genes central to glucose homeostasis, as was seen in a juvenile rat model of ZnD. We therefore speculate that ZnD alters gene expression through epigenetic mechanisms.