The polymorphic microsatellite in the proximal SLC11A1 promoter directs transcriptional regulation by HIF-1α. (A) Luciferase activities from 100 ng SLC11A1-luc constructs in both sense and antisense (SLC11A1-lucA) orientations compared with the construct -137-luc, which lacks the polymorphic microsatellite. Fold activation was with respect to pGL3Basic activity. (B) Comparison of SLC11A1-luc transcriptional activation by HIF-1α and HIF-2α. (C) HIF-1α regulation of SLC11A1 promoter activity is orientation independent and requires the polymorphic microsatellite. Luciferase expression was determined for promoter constructs transfected without or with 100 ng HIF-1α; fold activation is a ratio of transactivation and basal luciferase activities. Empty pcDNA3.1 vector was added where necessary to ensure equivalent amounts of plasmids. Activity of -137-luc is in comparison with SLC11A1-luc. For A-C, data are means (±SEM). (D) Duplex microsatellite oligonucleotide constructs with intact HREa and HREb (NR1HREab-luc) or where either or both HREs were mutated (mutations are shown in lowercase) to generate NR1HREa-luc, NR1HREb-luc, and GT₂₂-luc, respectively; duplexes were subcloned into pGL3Promoter vector (see Table S1). (E) Transactivation of HRE-luciferase constructs by HIF-1α. The constructs (100 ng each) from panel D were transfected alone or with 100 ng HIF-1α plasmid into BHK cells. Luciferase activities were normalized to β-galactosidase internal control. Data are representative of 5 experiments (means ± SEM). (F) HIF-1α–deficient cells cannot support SLC11A1 expression. SLC11A1-luc (100 ng) was transfected alone or with 100 ng HIF-1α plasmid into wild-type (C4.5) and HIF-1α–deficient (Ka13.5) cells; luciferase activity was normalized to β-galactosidase levels. A vertical line separates Ka13.5 cells trans-complemented with HIF-1α by cotransfection. Data are representative of 5 independent experiments (means ± SEM).