Fig 3. Aop and Foxo broker transcriptomic outcomes with Pnt.

(A) In vitro (Drosophila S2 cells) AOP^ACT counteracts activation by PNT^P1 of a synthetic promoter containing EBMs, upstream of an Adh-Firefly^luciferase reporter. Activity is shown following normalisation to internal Renilla^luciferase controls, and calculation of fold-change over median expression in the absence of PNT^P1 and AOP^ACT. Promoter activation was subject to a significant AOP^ACT:PNT^P1 interaction (linear model F_1,16 = 41.725, p = 7.9e-6) (B-C) Foxo and Aop^ACT coordinate transcription by jointly countering Pnt's transcriptional output. Heatmaps show per-gene Z-score of log₂ fold-change expression (DESeq2 output) upon RU₄₈₆ feeding relative to controls. Rows represent all genes in the union of targets identified in flies bearing S₁06 transgenes along with UAS-Foxo, UAS-Aop^ACT, UAS-Pnt^P1, or combinations thereof. Gene sets are defined by pattern of response to RU₄₈₆ amongst genotypes. Set assignments shown by coloured side bar. (D-E) Aop^ACT, Foxo and Pnt^P1 interact to establish transcriptional programs corresponding to lifespan. For gut and fat body, plots show coordinates of samples on the first two dimensions of PCA for the genes whose expression was responsive to the combinatorial, interactive effects of the three TFs. Key shows samples’ groupings by previously-published lifespan outcomes[4] resulting from TF induction in the gut and fat body or the gut alone (noting that lifespan effects of combined Aop^ACT and Pnt^P1 expression are not known).