NFE2 positively regulates JMJD1C by binding regulatory sites. (A-C) Transactivation of the JMJD1C promoter by NFE2. (A) Schematic illustration of the JMJD1C reporter gene constructs. Open circles: predicted NFE2-binding sites as depicted in Figure 1A. The degree of phylogenetic conservation is illustrated. (B) Luciferase reporter vectors were cotransfected into HEK-293T cells with expression plasmids encoding either NFE2 and/or MafG. Data were normalized to the cotransfection with MafG alone, set as 1. Graphs represent mean ± SEM of 4 independent experiments. *P < .05 by Student t test. (C) Potential NFE2-binding sites were disrupted by site-directed mutagenesis in HEK-293T, indicated by crossed circles. Sequences of wt and mutated NFE2 sites are shown (altered bases in bold). Luciferase assays were performed as in panel B. Data were normalized to the −120-bp wt construct cotransfected with MafG only, set as 1. Graphs show mean and SEM of at least 3 independent experiments. ***P < .001, **P < .01 by 1-way ANOVA with the post hoc Tukey comparison test. (D) The effect of NFE2 silencing on JMJD1C expression. HEL cells were transduced with a lentivirus encoding an shRNA against NFE2 (sh) or a scrambled control (scr), sorted for GFP positivity, and analyzed by western blotting. The blot is representative of 3 independent experiments.