Misexpression of CBP and CBP^Hatmut have different effects on the Wg target Dll. Confocal images of late third-instar imaginal discs containing P[Dpp-Gal4] and either P[UAS-CBP] reared at 18°C (A–C) or P[UAS-CBP^Hatmut] reared at 29°C (D–F) stained for Wg (green) or Dll (red). Arrows indicate the location where the Dpp expression domain intersects the Wg D/V stripe. Although CBP expression causes a reduction of Wg expression in approximately half of the discs, the Dll pattern remains unchanged. In contrast, the large majority of discs expressing CBP^HATmut had normal or slightly expanded Wg expression and reduction of Dll expression in the Dpp expression domain.

Arm interacts with CBP in vitro and in vivo. (A) Western blot with anti-CBP antisera demonstrating that GST-Arm, but not GST alone, is able to pull down fly CBP from extracts of 293 cells expressing CBP. (B) Arm and fly CBP interact when overexpressed in Kc cells. Cells were transfected with plasmids expressing V5-tagged Arm^* and Flag-tagged CBP. Forty-eight hours post-transfection, cell extracts were prepared and immunoprecipitated with V5, Flag and control IgG antibodies and the precipitates were analyzed by Western blot with anti-V5 antibody. Flag precipitation pulled down a significant portion of Arm^*-V5, compared with control IgG. (C) Endogenous Arm and CBP interact. Kc cells were treated with WCM or control media for 4 h before extract preparation. Proteins were immunoprecipitated with anti-CBP antisera or control IgG and Western blot were analyzed with anti-Arm antibody. CBP interacts with endogenous Arm in Wg-stimulated cells. (D) Arm and CBP interact directly in vitro. Bacterially expressed GST or GST–CBP fragments of the indicated residues were incubated with ³⁵S-Met-labeled Arm C-terminal fragment (residues 429–815). Precipitated proteins were analyzed together with 5% of the input material by SDS–PAGE and autoradiography. Three CBP fragments bound to the C-terminal fragment of Arm, while fragments comprising residues 1200–2550 show similar binding as the GST-negative control. All experiments shown were performed multiple times, with similar results obtained.

CBP is required in the wing imaginal discs for Wg signaling. Confocal images of late third-instar wing imaginal discs. (A–H) nej³ mutant clones stained for the clonal marker GFP (A, E), Wg (B, F), Dll (C) and Sens (G). (A–D) In this CBP mutant clone, Wg expression is upregulated but expression of the Wg target Dll is greatly reduced (white arrows). (E–H) In this CBP mutant clone, Wg expression is normal but the Wg target Sens in not expressed. (I–L) P[En-Gal4]/P[UAS-CBP^RNAi] wing disc stained for Wg (I), Sens (J) and Dll (K). The En expression domain is to the right of the white arrows. Wg expression is unaffected by CBP depletion but Sens and Dll expression are greatly reduced.

p300 binds to the HMG domain of TCF4E and CBP/p300 represses Wnt signaling in human cells. (A) The TCF4E HMG domain directly binds to p300. Bacterially expressed GST or GST-TCF4 (HMG domain; residues 326–396) fusion protein were incubated with a radiolabeled fragment of human p300 (residues 1254–1899). After pull down and washing, proteins retained by GST or GST-Arm were analyzed together with 5% of the input material by SDS–PAGE and autoradiography. (B) Human SW480 cells were transfected with the either the Topflash or Fopflash reporters, along with increasing amounts (100–500 ng) of pSuper-CBP^RNAi or pSuper-p300^RNAi constructs (empty pSUPER vector was used to normalize the amount of DNA transfected), which express short hairpins corresponding to each gene. CBP or p300 depletion increases TOPFLASH but not FOPFLASH reporter activity. Transfected cells were assayed for luciferase as described in Figure 3 legend and Materials and methods.

Misexpression of CBP and a CBP^HATmut inhibit Wg target gene expression. (A, B) Micrographs of adult wings containing the P[C96-Gal4] driver and either P[UAS-lacZ] (A) or P[UAS-CBP] (B). Misexpression of CBP leads to loss of wing margin (arrows in (B)). Expression of CBP^Hatmut produced a similar phenotype (data not shown). (C–K) Confocal images of late third-instar wing imaginal discs stained for Wg (green) or the Wg target Sens (red). (C–E) P[Dpp-Gal4]/P[UAS-CBP] disc reared at 18°C. Mild defects in Wg and Sens expression are sometimes observed. (F–H) P[Dpp-Gal4]/P[UAS-CBP] disc reared at 25°C. Most of the discs have a loss of Wg and reduction of Sens in the Dpp expression domain. (I–K) P[Dpp-Gal4]/P[UAS-CBP^Hatmut] disc reared at 29°C. The majority of the discs have no effect on Wg expression and a strong reduction in Sens expression.

CBP augments Arm transcriptional activity in a context-dependent manner. (A) 293 HEK cells were transfected with plasmids expressing an activated form of Arm (Arm^*; 20 ng) and fly CBP (200 or 400 ng) along with the Topflash luciferase reporter. CBP increases Arm activation of the Wnt reporter. (B) Fly Kc cells were transfected with Arm^* (20 ng) and CBP (50, 100, 200 or 400 ng) expression constructs along with the Dropflash luciferase reporter gene. CBP had a slight inhibitory effect on the ability of Arm^* to activate the Wg reporter. (C–E) Kc cells transfected with plasmids (20 ng) expressing the Gal4-DBD domain alone or Gal4-DBD fused to full-length Arm (Gal4-Arm), the N-terminal half of Arm (Gal4-ArmN; residues 1–428) or the C-terminal half of Arm (Gal4-ArmC; residues 429–815). CBP or CBP^Hatmut constructs were transfected as indicated at 20 or 50 ng. The Gal4 UAS-luciferase (UAS-luc) reporter gene was activated by Gal4-Arm, Gal4-ArmN and Gal4-ArmC, and CBP coexpression augmented this activation for Gal4-Arm and Gal4-ArmC but not Gal4-ArmN. CBP^HATmut did not effect Gal4-ArmC activation of UAS-luc. All transfections contained a lacZ expression plasmid, and luciferase activities were determined 48 h post-transfection and normalized against β-galactosidase activity. Values are the mean of duplicate experiments (standard deviations are indicated) and expressed as relative activity compared with cells transfected with the reporter alone.

CBP is required for the transcriptional activation of Wg endogenous targets genes in Kc cells. (A–E) Cells were treated for 4 days with control dsRNA (10 μg/well) or different doses (7.5 and 10 μg/well) of dsRNA corresponding to CBP. Cells were then incubated for 5 h with control or WCM before transcript levels of nkd (A), notum (B), α-tubulin (C), arm (D) and TCF (E) were measured by quantitative RT–PCR as described in Materials and methods. Results for nkd and notum were normalized to the average of α-tubulin, arm and TCF expression, while the later three were normalized to total RNA. Wg activation of nkd and notum were reduced in CBP-depleted cells. (F) Western blot showing that induction of Arm protein and TCF protein levels were not affected by CBP depletion.

CBP is recruited to the WRE of the nkd gene in a Wg- and Arm-dependent manner. (A, B) ChIP using antibodies against TCF (A) and Arm (B) demonstrate enhanced binding to a cluster of TCF binding sites in the nkd intron (compared with the nkd ORF) in Kc cells stimulated with WCM for 4 h. (C) Kc cells were transfected with an Arm^* expression plasmid and an hsp70 luciferase reporter containing a 420 bp fragment of the nkd intron (nkd-luc) or the same sequence with five predicted five TCF destroyed by site-directed mutagenesis (nkdmut-luc). Reporter gene activity was assayed as described in Figure 3 and Materials and methods. Arm^* activated nkd-luc 30-fold but did not activate nkdmut-luc. (D) CBP is recruited to the nkd WRE in a Wg and arm-dependent manner. Cells were transfected with control or arm dsRNA and cultured for 4 days before treatment with control or Wg-CM for 4 h before lysis and ChIP analysis with anti-CBP antisera. Precipitated DNA were purified and detected by Q-PCR using primers specifically against the nkd WRE or ORF as described in Materials and methods. Values are the mean of duplicate precipitations (±standard deviations) and the data is expressed as percentage of input DNA.