Fig. 2.

ETS2 directly regulates the expression of miR-196b. (A) ChIP assays with anti-Flag and anti-ETS2 antibodies showed binding of Flag-ETS2 to the promoters of miR-196b in AGS cells transfected with Flag-ETS2. Dual specificity phosphatase 6 (DUSP6) and aurora kinase A (AURKA) are confirmed ETS2 targets genes (46,47) and used as positive controls. The regions amplified by ChIP are: miR-196b-1: −851 to −683, miR-196b-2: −533 to −159 and miR-3: −154 to −3. I, input DNA; α-F, immunoprecipitation with anti-Flag antibody from cells expressing Flag-ETS2; α-E, immunoprecipitation with anti-ETS2 antibody from cells expressing Flag-ETS2; α-mIgG, immunoprecipitation with mouse IgG antibody was used as a negative control. (B) The promoter of miR-196b was repressed by ETS2. 293T cells were transiently cotransfected with Flag-ETS2 or mock vector and the wild-type miR-196b promoter (FL) or the various truncated miR-196b promoter constructs: N, C and P. These promoter sequences included in each were indicated by the numbers at the 5′ and 3′ termini. Normalized luciferase activity from triplicate samples is presented relative to that of cells transfected with the pGL3-basic construct. (C) The mutation of ETS2-binding motifs induced the promoter activities of miR-196b. 293T cells were transiently cotransfected with Flag-ETS2 or mock vector and wild-type miR-196b promoter (FL) or miR-196b promoter ETS2 binding site mutants (M1, M2 and M3). The miR-196b promoter constructs with the mutated core binding sequences were designated as M1, M2 or M3. Normalized luciferase activity from triplicate samples is presented relative to that of cells transfected with the FL construct. Luciferase activity was measured 48 h after transfection. The transfection efficiency was normalized against pRL-TK activity. The experiment was repeated twice with similar results. *P < 0.05, **P < 0.01.