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High doses of zinc oxide are commonly used in weaned pig diets to improve performance and health. Recent reports show that this may also lead to an imbalanced zinc homeostasis in the animal. For a better understanding of the regulatory mechanisms of different zinc intakes, we performed a feeding experiment to assess potential epigenetic regulation of the  ZIP4  gene expression via DNA methylation in the small intestine of piglets. Fifty-four piglets were fed diets with 57 (LZn), 164 (NZn) or 2,425 (HZn) mg Zn/kg feed for one or four weeks. The  ZIP4  expression data provided significant evidence for counter-regulation of zinc absorption with higher dietary zinc concentrations. The CpG +735 in the second exon had a 56% higher methylation in the HZn group compared to the others after one week of feeding (8.0·10 -4  <  p  < 0.035); the methylation of this CpG was strongly negatively associated with the expression of the long  ZIP4  transcripts ( p  < 0.007). In the LZn and NZn diets, the expression of the long  ZIP4  transcripts were lower after four  vs . one week of feeding (2.9·10 -4  <  p  < 0.017). The strongest switch leading to high DNA methylation in nearly all analysed regions was dependent on feeding duration or age in all diet groups (3.7·10 -10  <  p  < 0.099). The data suggest that DNA methylation serves as a fine-tuning mechanism of  ZIP4  gene regulation to maintain zinc homeostasis. Methylation of the  ZIP4  gene may play a minor role in the response to very high dietary zinc concentration, but may affect binding of alternate zinc-responsive transcription factors.

The Zinc Concentration in the Diet and the Length of the Feeding Period Affect the Methylation Status of the ZIP4 Zinc Transporter Gene in Piglets