Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling

Abstract

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.

Figure 1.

Coop and Pan interact with each other and form a complex on DNA. (A–C) In vitro translated samples were used for GST pull-down. (A) Control Luciferase and full-length Pan (lanes 1,2, 15% input) were pulled down by GST (lanes 3,4) or GST-Coop (lanes 5,6). (B) Control Luciferase and Coop (lanes 1,2, 5% input) were pulled down by GST (lanes 3, 4), GST-Pan (1–160) (lanes 5,6), or GST-Pan (247–362) (lanes 7,8). (C) Control Luciferase, CoopN (1–160), and CoopC (161–358) (lane 1, 5% input) were pulled down by GST (lane 2) or GST-Pan (247–362) (lane 3). (D) Kc cells were transfected with Flag-tagged Pan, or cotransfected with Flag-tagged Pan and HA-tagged Coop. Coop was immunoprecipitated by anti-HA antibody-conjugated beads and was analyzed by Western blotting. (E) Pan (247–362) caused a mobility shift (arrow) of a DNA probe harboring a Pan-binding site (PBS), but did not affect the mobility of a DNA probe harboring a mutated Pan-binding site (PBS*). Free probes are indicated by the arrowhead. (F) Coop alone did not bind to a DNA probe harboring a PBS, but caused a supershift in the presence of Pan (arrow).

Figure 2.

Overexpression of Coop represses Wg target Dll in a dosage-dependent manner. (A–A″) The expression of apGal4 alone did not affect Dll expression. (B–B″) The expression of Coop by apGal4 abolished Dll expression. (C–C″) The expression of enGal4 did not affect Dll expression. (D–D″) The expression of Coop by enGal4 strongly reduced Dll expression. Bar, 100 μm.

Figure 3.

Coop is not a universal repressor. (A–D) The expression of Coop by apGal4 strongly repressed Dll, but had no effect on lgs (A′), dpp (B′), omb (C′), or sal (D′) expression. Dll staining is in green, and staining of Lgs or the lacZ reporters is in red. Bar, 100 μm.

Figure 4.

Loss of Coop enhances expression of Dll in vivo. (A) Expression of Dll in wild-type control. (B) Expression of dsRNA targeting Coop mRNA by enGal4-enhanced Dll expression. (C) Expression of Dll was enhanced in coop clones induced by hs-flp; FRT42 ubi-GFP Minute/FRT42 coop^R62. Bar, 100 μm.

Figure 5.

Coop and Arm compete for Pan binding. (Left panels) Presence of transfected Pan, Arm, and Coop are shown by Western blotting. (A, right panels) Coop reduced the amount of Pan coimmunoprecipitated along with Arm. (B, right panels) Arm reduced the amount of Pan coimmunoprecipitated along with Coop.