The Krüppel-like zinc finger protein Glis2 functions as a negative modulator of the Wnt/beta-catenin signaling pathway

Abstract

To gain insight into the mechanism by which Gli-similar 2 (Glis2) regulates transcription, we performed yeast-two hybrid cDNA library screening using Glis2 as bait. This screening identified beta-catenin as a potential Glis2-interacting protein. Mammalian two-hybrid, co-immunoprecipitation, and GST-pulldown analyses supported the interaction between Glis2 and beta-catenin. Pulldown analyses with several Glis2 deletion mutants indicated that the 1st zinc finger motif of Glis2 is critical for its interaction with beta-catenin, while the armadillo repeats of beta-catenin are important in its interaction with Glis2. Reporter analyses showed that Glis2 represses T-cell factor (TCF)-mediated transcriptional activation. In addition, Glis2 represses the expression of the TCF target gene cyclin D1. Our results indicate that Glis2 interacts with beta-catenin and suggest that Glis2 functions as a negative modulator of beta-catenin/TCF-mediated transcription.

Fig. 1

(A) Identification of β-catenin as a Glis2-interacting protein by mammalian two-hybrid analysis. HCT116 cells were co-transfected with (UAS)₅-LUC, pRL-SV40, pM, pVP16, pM-Glis2, and increasing amounts of pVP16-β-catenin as indicated. After 48 h, cells were harvested and assayed for reporter activities. The relative LUC activity was calculated and plotted.

Fig. 2

Glis2 and β-catenin are associated with the same protein complex. (A) HCT116 cells were transfected with p3xFlag-CMV-Glis2, p3xFlag-CMV, and pEGFP-wt-β-catenin as indicated. Forty-eight h after transfection, protein lysates were prepared and one part was used in Western blot (WB) analysis with an anti-GFP (upper panel) or anti-Flag M2 antibody (middle panel). The remaining was used for immunoprecipitation (IP) using anti-Flag M2 affinity resin. Bound proteins were then examined by Western blot analysis with anti-GFP antibody (lower panel). (B) Glis2 interacts with endogenous β-catenin. HCT116 cells were transfected with p3xFlag-CMV-Glis2 or p3xFlag-CMV. Forty-eight h after transfection, cell lysates were prepared and Flag-Glis2 protein complexes immunoprecipitated with anti-Flag affinity resin. Bound proteins were examined by Western blot analysis with anti-β-catenin or anti-Flag antibodies. (C) The interaction of β-catenin is specific for Glis2. HCT116 cells were transfected with V5-β-catenin, p3xFlag-CMV, p3xFlag-CMV-Glis1, or p3xFlag-CMV-Glis2. Proteins in the cellular lysates and immunoprecipitated proteins were examined by Western blot analysis using anti-Flag M2 or anti-V5 antibodies.

Fig. 3

Glis2 interacts directly with β-catenin. (A) Interaction of GST and GST-β-catenin fusion proteins with [³⁵S]-methionine-labeled Glis2 or Glis1 was examined as described in Materials and Methods. First lane, 10% input; G, GST; C; GST-β-catenin. Arrow indicated Glis1 and arrow head indicated Glis2. (B) Glis2 interacts with β-catenin through its ZFD. GST-pulldown analysis with [³⁵S]-methionine-labeled Glis2, Glis2(1–169), Glis2(170–520), Glis2(324–520) or luciferase which served as negative control. Bound radiolabeled proteins were separated by PAGE and visualized by autoradiography. (C, D) Co-immunoprecipitation assay using Glis2 mutants. HCT116 cells were transfected with V5-β-catenin, p3xFlag-CMV empty vector, p3xFlag-CMV-Glis2, p3xFlag-CMV-Glis2(1–382), p3xFlag-CMV-Glis2(1–200), p3xFlag-CMV-Glis2(1–143), p3xFlag-CMV-Glis2(170–377), or p3xFlag-CMV-Glis2ZF1^mut containing the C175A mutation in ZF1 (ZF1^mut). Proteins in the cellular extracts and immunoprecipitated proteins were examined by Western blot analysis using anti-Flag M2 or anti-V5 antibodies.

Fig. 3

Glis2 interacts directly with β-catenin. (A) Interaction of GST and GST-β-catenin fusion proteins with [³⁵S]-methionine-labeled Glis2 or Glis1 was examined as described in Materials and Methods. First lane, 10% input; G, GST; C; GST-β-catenin. Arrow indicated Glis1 and arrow head indicated Glis2. (B) Glis2 interacts with β-catenin through its ZFD. GST-pulldown analysis with [³⁵S]-methionine-labeled Glis2, Glis2(1–169), Glis2(170–520), Glis2(324–520) or luciferase which served as negative control. Bound radiolabeled proteins were separated by PAGE and visualized by autoradiography. (C, D) Co-immunoprecipitation assay using Glis2 mutants. HCT116 cells were transfected with V5-β-catenin, p3xFlag-CMV empty vector, p3xFlag-CMV-Glis2, p3xFlag-CMV-Glis2(1–382), p3xFlag-CMV-Glis2(1–200), p3xFlag-CMV-Glis2(1–143), p3xFlag-CMV-Glis2(170–377), or p3xFlag-CMV-Glis2ZF1^mut containing the C175A mutation in ZF1 (ZF1^mut). Proteins in the cellular extracts and immunoprecipitated proteins were examined by Western blot analysis using anti-Flag M2 or anti-V5 antibodies.

Fig. 4

β-catenin interacts with Glis2 through its specific regions. GST and several GST-β-catenin fusion proteins were bound to glutathione-Sepharose 4B beads and then incubated with [³⁵S]-methionine-labeled Glis2. Bound radiolabeled proteins were visualized by autoradiography. The input of the various GST-β-catenin proteins is shown in the lower panel. Asterisks indicate the respective GST-β-catenin proteins.

Fig. 5

Glis2 enhances nuclear localization of β-catenin. NEH293 cells were transiently transfected with p3xFlag-CMV-Glis2, p3xFlag-CMV-Glis2ZF1^mut, and/or pEGFP-β-catenin. The percentage of cells in which the localization of Glis2 or β-catenin was restricted to the nucleus, was calculated and plotted. The number of cells counted is indicated above each bar.

Fig. 6

Glis2 represses TCF-mediated transcriptional activity and the induction of cyclin D1 by β-catenin. (A) HCT116 cells were cotransfected with TOPFlash and FOPFlash reporters, pCMVbeta, β-catenin(ΔN90), and p3xFlag-CMV-Glis2 as indicated. Forty-eight h after transfection, cells were assayed for reporter activities as described in Materials and Methods. (B) HCT116 cells were cotransfected with Super8XTOPFlash and Super8XFOPFlash reporters, pRL-SV40, pCMV-V5-β-catenin (β-cat(S37C)) and different concentrations of p3xFlag-CMV-Glis2 expression vector as indicated and then processed as described under A. (C) HCT116 cells were cotransfected with the cyclin D1 reporter PCD1, pRL-SV40, the constitutively-active pCMV-V5-β-catenin (S37C) expression vector, and p3xFlag-CMV-Glis2 as indicated and then processed as described under A. (D) HCT116 cells were transfected with pCMV-V5-β-catenin and p3xFlag-CMV-Glis2 (1.0 and 1.5 μg) as indicated. Thirty-six h later, cellular proteins were analyzed by Western blot analysis using antibodies against V5, Flag, β-tubulin, and cyclin D1. The lower molecular weight band (arrow head) in the Flag-Glis2 Western blot represents a proteolytically processed form of Glis2.

Fig. 6

Glis2 represses TCF-mediated transcriptional activity and the induction of cyclin D1 by β-catenin. (A) HCT116 cells were cotransfected with TOPFlash and FOPFlash reporters, pCMVbeta, β-catenin(ΔN90), and p3xFlag-CMV-Glis2 as indicated. Forty-eight h after transfection, cells were assayed for reporter activities as described in Materials and Methods. (B) HCT116 cells were cotransfected with Super8XTOPFlash and Super8XFOPFlash reporters, pRL-SV40, pCMV-V5-β-catenin (β-cat(S37C)) and different concentrations of p3xFlag-CMV-Glis2 expression vector as indicated and then processed as described under A. (C) HCT116 cells were cotransfected with the cyclin D1 reporter PCD1, pRL-SV40, the constitutively-active pCMV-V5-β-catenin (S37C) expression vector, and p3xFlag-CMV-Glis2 as indicated and then processed as described under A. (D) HCT116 cells were transfected with pCMV-V5-β-catenin and p3xFlag-CMV-Glis2 (1.0 and 1.5 μg) as indicated. Thirty-six h later, cellular proteins were analyzed by Western blot analysis using antibodies against V5, Flag, β-tubulin, and cyclin D1. The lower molecular weight band (arrow head) in the Flag-Glis2 Western blot represents a proteolytically processed form of Glis2.