The role of MREs in the mouse FPN1 promoter. (A) Diagram of the mouse FPN1 promoter shows 2 putative MREs at positions −990 (tgcaccc) and −879 (tgcactc on the reverse strand) from the TATAA box. Plasmids were constructed containing portions of FPN1 promoter region in front of a Firefly luciferase reporter. The areas of the FPN1 promoter contained regions −2378, −1154, or −623 to the TATAA box lacking the IRE region (called p2500, p1500, p1000). Macrophages or NIH3T3 cells were transfected with p2500, p1500, and p1000 plasmids and subsequently treated with 100μM zinc or 100μM FAC. Cells were also transfected with a Renilla luciferase containing plasmid to control for transfection efficiency. Sixteen hours later, cells were assayed for luciferase activity. Data presented are expressed as percentage of Firefly luciferase light units that have been normalized using the Renilla luciferase. (B) Chromatin immunoprecipitations were performed on MTF-1-Flag expressing NIH3T3 cells exposed to zinc, iron or no metal using antibodies against Flag as described in “Chromatin immunoprecipitation.” (C) The putative MREs in the p2500 plasmid were mutagenized, the plasmid transfected into cells, and cells incubated with cobalt or cadmium and luciferase assayed after 16 hours. All experiments were performed a minimum of 3 times.