region and two insulin-responsive elements (IREs) at the middle and distal regions within −3 kb of TH promoter (Fig. 7d)39. We therefore performed chromatin immonoprecipitaion (ChIP) assays to determine whether FoxO1 binds directly to these potential binding sites. Both in vivo and in vitro ChIP assays using whole-brain and Neuro2A cells transfected with myc-tagged FoxO1-ADA, confirmed a specific and direct binding of FoxO1 on the conserved FoxO1-binding sequence at the proximal region of TH promoter (Fig. 7d). The binding of FoxO1 on the IREs in the middle and distal regions of TH promoter might not be specific as the ChIP signals were also observed with the bead and negative IgG control (Supplementary Fig. 6d). Based on the ChIP analyses, we next established luciferase constructs containing TH promoter regions with or without FoxO1 potential binding sites and measured the promoter activity (Fig. 7e). Overexpression of FoxO1 (WT FoxO1) significantly suppressed the luciferase activity of the constructs containing the potential binding sites and this suppression of FoxO1 on TH promoter was more pronounced in constitutive active FoxO1 (ADA FoxO1; Fig. 7e). In contrast, the promoter activity of TH gene was significantly increased after suppression of endogenous FoxO1 by overexpression of dominant-negative form
