Deletion Analysis of the NF-E2 1A promoter in K562 and UKE-1 cells. A DNA fragment encoding bp −3606 to bp +1 upstream of the exon 1A transcriptional start site was cloned into the pGL3-Luciferase reporter vector. A deletion mutant encoding bp −1740 to +1 of the NF-E2 1A promoter was generated. Both constructs as well as the empty pGL 3 luciferase vector were transiently nucleofected into K562 cells (A) or UKE-1 cells (B). At 24 hours after transfection, luciferase activity was determined. Results were normalized for transfection efficiency by cotransfection of a Tk-driven Renilla luciferase reporter gene vector. Mean and standard deviation of 3 independent experiments, each measured in duplicate, are shown. ***P < .001, one-way ANOVA, *P < .05 Kruskal Wallis one-way ANOVA on Ranks. (C) Phylogenetic conservation of the NF-E2 1A promoter region. Top: schematic representation of the NF-E2 promoter, exon 1A, indicated by an open box, and the 3 predicted AML1 binding sites, indicated by black boxes and numbered I, II, and III. Top row: Multiple alignment of the NF-E2 promoter sequence across all indicated species by use of Genome Vista software. Promoter segments showing more than 75% identity are highlighted in pink, exonic sequences in cyan. Remaining rows: Pair-wise alignment of the NF-E2 promoter species across the indicated species. Hs indicates Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; cf, Canis familiaris; Ec, Equus caballus; Pa, Pongo pygmaeus abelii; and Pt, Pan troglodytes. Promoter segments showing more than 75% identity are highlighted in pink, exonic sequences in cyan.