Deletion mapping of the human hepcidin gene promoter. (A) HepcP1.1-luc (wild-type [WT]) was selectively restricted with enzymes to remove various segments. Constructs were generated by restriction with BalI(ΔBal; –598 to –379), PmlI(ΔPml; –526 to –454), NheI-AflII (ΔNA; –972 to –821), AflII-PmlI(ΔAP; –821 to –454), NheI-PmlI (ΔNP; –972 to –454), and NheI-TthIIII (ΔNT; –972 to –196). Nucleotide numbering is based on Courselaud et al.²¹  Note that the NheI restriction site was used for cloning purposes only and is not intrinsic to this segment of the promoter (PCR primers in “Materials and methods”). Also, although there are 2 TthIIII sites within the promoter, double-digestion with NheI removed all but approximately 200 bp of the promoter. (B) Basal transcriptional activity of promoter deletion constructs from panel A. Constructs (100 ng each) were cotransfected with pSVβgal (50 ng) into BHK cells for 48 hours, and reporter activities were measured as described above. Luciferase levels were normalized with respect to βgal activity. Fold activation was based on the activity of pGL3Basic, and assigned an arbitrary activation level of 1. Pairwise comparisons were made between WT and deletion constructs using the 1-way ANOVA/Student-Newman-Keuls test. **P < .005. Data are representative of 4 independent sets of transfections, shown as means ± SEM.