Figure 3: Yki directly regulates dilp8 expression through a HRE in the dilp8 promoter.

(a) Schematic of the dilp8 promoter region and the HRE harbouring the three putative Sd-binding sites (indicated as green squares). The dilp8 promoter fragments used to study Yki-dependent regulation of dilp8 expression in vivo are shown. In dilp8-PFΔ123, mutations of the three putative Sd-binding sites are indicated as red squares. (b) DNA pull-down experiments show that binding of Sd to the dilp8-PF is mediated by the three Sd-binding sites. The indicated DNA fragments were incubated with lysates from S2 cells transfected with Sd-Flag. Band intensities represents the average of three independent experiments: for diap-PF: 3.8±0.7 (positive control), for act: 1±0.2 (negative control), for dilp8-PF: 5.1±1.0, and for dilp8-PF Δ123: 1.9±0.4. (c) Luciferase assay showing that Yki/Sd activate gene expression through the HRE in the dilp8-PF. S2 cells were transfected with Yki and Sd. The ability of Yki/Sd to induce gene expression from the indicated promoter fragments was measured (triplicate samples, error bars represent s.e.m.). (d–s) Yki induces dilp8 transcription through the HRE in vivo. Wing imaginal discs carrying GFP-labelled Yki-expressing clones were dissected from transgenic flies carrying the indicated dilp8 promoter fragments fused to the LacZ-encoding sequence. The full dilp8 promoter and dilp8-PF, but not dilp8-intron1 and dilp8-PFΔ123, induces lacZ expression as detected by β-gal staining (d,h,l,p in red) in the GFP-labelled yki-overexpressing clones (e,i,m,n in green). In each condition, yki overexpression leads to elevated levels of endogenous Dilp8 protein (f,j,n,r in white).