Figure 5.

dFOXO directly activates transcription of dInR and d4EBP. (A) Luciferase assays showed activation of d4EBP and dInR promoters in S2 cells after cotransfection with dFOXOA3 (white bars). (Black bars) Empty vector. (B) Band shift performed with d4EBP and dInR promoters showed that recombinant dFOXO binds specifically to these promoters. (C) dFOXO binds specifically to d4EBP and dInR promoters in vivo. ChIP of cross-linked extracts of S2 cells expressing dFOXO or dFOXOA3 grown in prescence of insulin. (D) Recombinant dFOXO activates transcription of d4EBP (lanes 1–4) and dInR (lanes 5–9) promoters and of a synthetic promoter bearing 4 FREs (lanes 10–13) in vitro. (E) Schematic representation of the d4EBP and dInR promoters showing the putative FREs (striped boxes) located upstream of the transcription start sites (indicated by arrows). The probes used in band shifts are indicated below as horizontal brackets, and those probes bound by dFOXO are shown as thick lines. Thick horizontal bars indicate the DNA regions bound in vivo by dFOXO as analyzed by ChIP.