The Kto-Skd complex can regulate ptc expression by interacting with Cubitus interruptus (Ci) in the Hedgehog signaling pathway

Abstract

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments that derive from adjacent cell populations. The anterior/posterior (A/P) boundaries, which are critical to maintaining the position of organizers, are established by a complex mechanism involving Hh signaling. Here, we uncover the regulation of ptc in the Hh signaling pathway by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, we provide further evidence that Kto-Skd affects the A/P-axial development of the whole wing disc. Kto can interact with Cubitus interruptus (Ci), bind to the Ci-binding region on ptc promoter, which are both regulated by Hh signals to down-regulate ptc expression.

Keywords: Development; Drosophila; Hedgehog Signaling Pathway; Signal Transduction; Transcription.

FIGURE 1.

Loss of function of both kto and skd enlarges the A compartment size and increases the width of the Ptc domain. Wing imaginal discs expressing UAS-GFP-ptc-lacZ alone (A–A‴) or combined with UAS-kto RNAi (B–B‴), UAS-skd RNAi (C–C‴), both UAS-kto RNAi and UAS-skd RNAi (D–D‴), UAS-Cdk8 RNAi (E–E‴), UAS-CycC RNAi (F–F‴) driven by the dorsal compartment-specific driver ap-Gal4 were stained with anti-Ci or LacZ antibody to show Ci (red) and LacZ signal (blue). The GFP signal marks gene expression regions. White arrows mark the boundary of dorsal and ventral compartments of the wing imaginal disc. a–d, magnified images show the signals of both Ci and LacZ. B–B‴, b, knockdown of kto resulted in Ci and Ptc expanding to the P compartment and Ptc expression region at the A/P boundary becoming wider with a slight up-regulated expression level compared with the wild type control (A–A‴, a). C–C‴, c, knockdown of skd induced the similar phenotype with knockdown of kto. D–D‴, d, wing disc with both kto RNAi and skd RNAi showed more obvious phenotypes than with either of the two gene RNAi. E–F‴ and e–f, wing imaginal discs with Cdk8 or CycC RNAi showed close to the wild-type phenotype.

FIGURE 2.

Kto-Skd complex can down-regulate the expression of ptc mainly in high levels of Hh. A–C‴, wing imaginal discs expressing UAS-GFP alone (A–A‴) or together with UAS-kto RNAi (B–B‴), UAS-skd RNAi (C–C‴) driven by AG4-Dicer2 were stained with anti-Ci (red) and anti-Ptc antibody (blue). GFP signals (green) label the clones in which target genes were knocked down. Arrows indicate clones along the A/P boundary both dorsal and ventral in which Ci and Ptc are up-regulated. a–c′, magnified images show the protein levels of Ci and Ptc. D, S2 cells were transfected with plasmids of UAS-kto, UAS-skd, or dsRNA of kto, respectively, on the basis of ptc-luciferase assay system. Overexpression of Kto or Skd suppressed ptc-promoter activity, especially in the presence of Hh, and knockdown of kto in S2 cells up-regulated the activity of ptc-luciferase reporter.

FIGURE 3.

Hh signal regulates the interaction between Ci and Kto or Skd. A–E, S2 cells were transfected with the indicated constructs and analyzed by Co-IP and Western blot. A and B, Myc-Kto and Myc-Skd interact with 3×FLAG-Ci (flg-Ci), and the interaction may be greatly enhanced by the treatment of Hh. C and D, S2 cells were co-transfected with Smo mutants, SmoSA, SmoWT, or SmoSD, to mimic differential Hh pathway activity. SmoSD, which mimics high level activity of the Hh pathway, dramatically enhances the interaction between 3×FLAG-Ci and Myc-Kto (C) or Myc-Skd (D). In contrast, SmoSA and SmoWT, which mimic none or low levels of Hh, cannot promote interaction of the proteins. E, HA-Kto, Myc-Skd, and 3×FLAG-Ci were together overexpressed in S2 cells for two-step Co-IP. HA-Kto and Myc-Skd may be precipitated in the first IP of 3×FLAG-Ci. HA-Kto and 3×FLAG-Ci may be precipitated in the second IP of Myc-Skd. These suggested Kto, Skd, and Ci could form a large complex.

FIGURE 4.

Kto and Ci interact with each other through their N-terminal parts. A, schematic drawings of Kto and Ci and their fragments. B–D, S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h transfection, lysates from transfected S2 cells were immunoprecipitated with anti-FLAG (flg) or anti-Myc agarose beads. Stars indicate the target proteins. B and C, N-terminal part of Kto interacted with Ci. D, Kto fragment (amino acids 290–1309) could bind to most parts of Ci.

FIGURE 5.

Kto and Ci can localize on the same ptc promoter locus. A, S2 cells were transfected with plasmids of UAS-kto, UAS-kto-a, or UAS-kto-b respectively on the basis of ptc-luciferase assay system. Overexpression of Kto or the mapped two fragments of Kto suppressed ptc-promoter activity, which indicates that the two Kto fragments are both suppressive domains. B, schematic drawings of Ci binding locus on the ptc promoter and the regions covered by the ChIP primers. C and D, S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were prepared for the ChIP assay. Data from ChIP signals were normalized to 1/10 of input and shown as the fold change to the first group (mean ± S.D.; n = 3).