Fig. 3.

AtEAM discovery and characterization. (A) Distribution analysis of the AtEAM shown as pie charts, including the absolute numbers for each category and percentile. The motif is shown on top. The plus sign indicates that the category includes the shown number of motifs or more. The majority of Atoh1-bound regions posses at least one AtEAM. (B) EMSA validating the Atoh1 binding efficiency to AtEAM; Neuro2a cells and labeled oligo (lane 1); transfected with Atoh1-FLAG (lanes 2–5) or Ascl1-FLAG expression plasmids (lane 6). Although there is a weak shift in the Ascl1-FLAG transfected sample (lane 6), Atoh1-FLAG transfection results in a strong shift (lane 2) compared with the untransfected control (lane 1). Competitors are in 100× molar excess. (C) Dual luciferase reporter assay using a firefly reporter construct harboring one AtEAM in front of a minimal promoter either WT or mutated in designated positions. Data are presented as mean ± SEM after normalization to Renilla control. Cotransfection of Atoh1-FLAG (250 ng) activates the WT reporter [0 mismatch mutations (MM), green bar] over twofold. One mismatch mutation (orange) had little effect on the induction capacity of Atoh1; 2 MM (red) abolished the activation. Mutation positions are indicated on the x axis. ANOVA with Tukey's post hoc t test revealed a statistical significance of *P < 0.01. (D) Dual luciferase reporter assay using WT luciferase construct in DAOY cells, cotransfected with either Atoh1-FLAG (green bars), Ascl1-FLAG (red bars), or Ngn1 (blue bars). Data are presented as mean ± SEM. Triangle indicative for increasing amounts of transfected DNA (0, 50, 100, 250 ng). Only Atoh1 is able to activate the reporter (*P < 0.01).