DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression

Production of the iron regulatory peptide hepcidin is tightly controlled by a network of proteins in hepatocytes that sense levels of iron in the circulation (as diferric-transferrin) and in tissues (in ferritin). Human studies show high variability in the normal range of serum hepcidin levels. We have postulated that this may, in part, be related to inter-individual variability in the expression of genes in the iron sensing pathway, potentially governed by epigenetic factors. Here, we have investigated whether genes encoding hepatic iron sensing proteins and hepcidin are regulated by DNA methylation. Experiments were performed on two human hepatoma cell lines, HepG2 cells and Huh7 cells. Basal expression of TFR2 and HAMP was significantly lower in Huh7 cells compared with HepG2 cells. Analysis of bisulphite-converted DNA from Huh7 cells revealed partial methylation of TFR2 (alpha transcript), which could result in gene silencing. Demethylation using 5-aza-2'-deoxycitidine (AZA) increased TFR2 mRNA expression in Huh7. PCR analysis of bisulphite-converted HAMP promoter DNA, using methylation-specific primers, revealed no differences between cell lines. However, HAMP mRNA expression in Huh7 was increased by AZA treatment, suggesting that methylation of one or more iron sensing genes may indirectly influence HAMP expression. Our study provides evidence that DNA methylation might control expression of HAMP and other hepatic iron sensing genes, and indicates that epigenetic influences on iron homeostasis warrant further investigation.