A highly conserved enhancer is responsible for the Stat3-mediated responsiveness of the miR-21 promoter to IL-6. (A) 130-bp regions containing 2 predicted Stat3 binding sites upstream of the miR-21 genes of various vertebrate species are aligned. T, G, C, and A nucleotides are colored green, orange, blue, and red, respectively. Stat3 sites are highlighted by yellow boxes. *Sequences from organisms where no genomic sequence was available were obtained from the Ensembl trace repository (Table S2). (B) XG-1 cells were deprived of IL-6 for 72 hours or restimulated for 30 minutes and subjected to a chromatin immunoprecipitation (ChIP) assay using anti-Stat3 or IgG isotype control. Coimmunoprecipitated DNA was amplified by PCR with primers specific for the miR-21 upstream enhancer. (C) Reporter gene assays were performed in HepG2 cells transfected either with a luciferase vector driven by the miR-21 promoter/enhancer alone (−) or in the presence of a vector encoding a small hairpin RNA silencing Stat3 expression (siStat3), or with a miR-21 promoter/enhancer construct containing point mutations in both Stat3 sites (ΔStat3). Values represent the mean luciferase activities ± SD of 3 independent experiments relative to samples from unmanipulated IL-6–treated cells.