The Kru¨Ppel-Like Zinc Finger Protein Glis2 Functions As a Negative

FEBS Letters 581 (2007) 858–864

The Kru¨ppel-like zinc ﬁnger protein Glis2 functions as a negative modulator of the Wnt/b-catenin signaling pathway

Yong-Sik Kim, Hong Soon Kang, Anton M. Jetten* Cell Biology Section, LRB, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

Received 3 November 2006; revised 19 December 2006; accepted 11 January 2007

Available online 2 February 2007

Edited by Ivan Sadowski

deacetylase 3 (HDAC3) [7]. However, the precise molecular Abstract To gain insight into the mechanism by which Gli-similar 2 (Glis2) regulates transcription, we performed yeast-two hy- mechanisms by which Glis2 regulate transcription are still brid cDNA library screening using Glis2 as bait. This screening poorly understood. identified b-catenin as a potential Glis2-interacting protein. To identify additional mediators or modulators of Glis2 Mammalian two-hybrid, co-immunoprecipitation, and GST-pull- activity, we performed yeast two-hybrid screening using the down analyses supported the interaction between Glis2 and b- amino terminus of Glis2 as bait. This analysis identified b-cate- catenin. Pulldown analyses with several Glis2 deletion mutants nin as a potential Glis2-interacting protein. Previous studies indicated that the 1st zinc finger motif of Glis2 is critical for have shown that b-catenin is a multifunctional protein [8– its interaction with b-catenin, while the armadillo repeats of b- 10]. The cadherin-bound form functions as a regulator of cell catenin are important in its interaction with Glis2. Reporter adhesion and migration while the cytoplasmic form functions analyses showed that Glis2 represses T-cell factor (TCF)-medi- as a transducer in the canonical Wnt signaling pathway. ated transcriptional activation. In addition, Glis2 represses the expression of the TCF target gene cyclin D1. Our results indi- Recent studies have indicated that b-catenin can also interact cate that Glis2 interacts with b-catenin and suggest that Glis2 with proteins other than those associated with the canonical functions as a negative modulator of b-catenin/TCF-mediated Wnt signaling pathway. These include several nuclear recep- transcription. tors, inhibitor of b-catenin and TCF-4 (ICAT), pontin, and Published by Elsevier B.V. on behalf of the Federation of reptin [11–13]. Moreover, several studies have provided evi- European Biochemical Societies. dence for a link between the hedgehog/Gli and Wnt/b-catenin signaling pathways [14,15]. Keywords: Gli-similar; Kru¨ppel-like zinc finger protein; In this study, we provide evidence for an interaction between b-Catenin; Gene expression Glis2 and b-catenin. The 1st zinc finger motif of Glis2 and the armadillo repeats of b-catenin are critical in the interaction between these two proteins. In addition, we demonstrate that Glis2 represses b-catenin/TCF-mediated transcription. Our observations indicate an interaction between the Glis2 and 1. Introduction b-catenin/TCF signaling pathways and suggest that Glis2 can function as a negative regulator of b-catenin/TCF-induced Gli-similar (Glis) 1–3 constitute a subfamily of Kru¨ppel-like gene expression. zinc finger proteins that are closely related to members of the Gli subfamily [1–5]. The highly conserved zinc finger domain mediates the binding of these proteins to Gli-binding sites (GBS), containing the consensus sequence GACCACCCAC, 2. Materials and methods in the regulatory region of target genes [2,5,6]. The Glis2 gene encodes a 56 kDa transcriptional regulator 2.1. Plasmids pEGFP-Glis2 was obtained by cloning full-length Glis2 into [4]. It is highly expressed in the ureteric bud and induced dur- pEGFP/C1. The sequences of all plasmids were verified by restriction ing neuronal differentiation suggesting that Glis2 is involved in enzyme analyses and DNA sequencing analyses. p3xFlag-CMV- the regulation of gene transcription at specific stages of kidney Glis2ZF1mut, containing a point mutation (C175A) in the first zinc fin- development and neurogenesis [3,4]. Its transcriptional activity ger (ZF1) of Glis2, was generated using a site-directed mutagenesis kit from Stratagene following the manufacturer’s instructions. For other is likely mediated through interactions with other nuclear pro- plasmids, see Supplements. teins that function as co-repressors or co-activators. This is supported by findings showing that the transcriptional repression by Glis2 is mediated through recruitment of the co-repres- 2.2. Immunoprecipitation (IP) assay and Western blot analysis Cells were transiently transfected with p3xFlag-CMV, p3xFlag- sors carboxyl-terminal binding protein 1 (CtBP1) and histone CMV-Glis2, pEGFP-b-catenin, V5-b-catenin, and truncated V5-b- catenin mutants as indicated, using Fugene 6 transfection reagent (Roche, Indianapolis, IN). After 48 h incubation, cells were harvested *Corresponding author. Fax: +1 919 541 4133. and lysed in RIPA buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, E-mail address: [email protected] (A.M. Jetten). 0.25% deoxycholate, 1% NP-40, 1 mM EDTA] containing protease inhibitor cocktails I and II (Sigma). Flag-Glis2 protein complexes were Abbreviations: Glis, Gli-similar; TCF, T-cell factor; TOP, TOPFlash; isolated using anti-Flag M2 affinity gel (Sigma). The bound protein FOP, FOPFlash complexes were solubilized in sample buffer and analyzed by Western

0014-5793/$32.00 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2007.01.058 Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864 859 blot analysis using mouse anti-Flag M2 (Sigma) or anti-GFP (Miltenyi taining proteins, the GTPase XPA binding protein 1 (XAB1) Biotec, Auburn, CA) antibodies. In certain cases proteins were immu- and G-protein signalling modulator 2 (GPSM2). Two indepen- noprecipitated with anti-b-catenin agarose resin (Santa Cruz Biotech- dently-derived b-catenin clones were isolated; one clone using nology, Santa Cruz, CA) and bound proteins examined by Western blot analysis with an anti-b-catenin (BD Pharmingen, San Jose, CA) a mixed human breast/prostate cancer cDNA library as prey, and anti-Flag M2 antibodies. To examine the regulation of cyclin the other from a mixed mouse embryo cDNA (E7-E19) D1 expression by Glis2, HCT116 cells were transfected with library. Further studies were carried out to analyze the puta- p3xFlag-CMV-Glis2 and V5-b-catenin expression vectors as indicated. tive interaction of Glis2 with b-catenin. Thirty-six hours later, cells were lysed directly in 2· sample buffer (Sig- ma). Protein lysates were examined by Western blot analysis with an The interaction between Glis2 and b-catenin was confirmed anti-Flag M2 antibody, and antibodies against V5 (Invitrogen), cyclin by mammalian two-hybrid analysis. HCT116 colorectal carci- D1, and b-tubulin (Santa Cruz Biotechnology). noma cells were co-transfected with (UAS)5-LUC reporter, pM or pM-Glis2 in the presence of increasing amounts of 2.3. In vitro pulldown assay pVP16-b-catenin. Co-expression of VP16-b-catenin signifi- Equal amounts of GST-b-catenin recombinant protein or GST were cantly increased reporter activity in a dose-dependent manner incubated with gluthathione–Sepharose 4B beads and washed in PBS. [35S]-methionine-labeled Glis2 was obtained using the TNT Quick due to the interaction of b-catenin with Glis2 (Fig. 1). Coupled Transcription/Translation system (Promega). The GST and In order to be physiologically relevant the two proteins need GST-b-catenin bound beads were then incubated with the [35S]-methi- to be co-expressed at least in some mammalian cells. There- onine-labeled Glis2 in 0.2 ml binding buffer (20 mM Tris–HCl, pH 7.6, fore, we examined the expression of Glis2 and b-catenin in 100 mM KCl, 0.05% Nonidet P-40, 0.1 mM EDTA, 10% glycerol and normal colon and several colorectal carcinoma cell lines (Sup- 1 mM PMSF). After 1 h incubation at 4 C, beads were washed five times in binding buffer and then boiled in 15 llof2· SDS–PAGE plement Fig. 1). Glis2 and b-catenin are both expressed in nor- loading buffer. Solubilized proteins were then separated by 4–20% mal colon and in Caco-2, HCT15, HCT116, Lovo, SW620, SDS–PAGE and the radiolabeled proteins visualized by autoradiogra- and SW480 cells. However, colorectal carcinoma Colo205 phy. and KM12 cells expressed only b-catenin. Next, we confirmed the interaction of Glis2 and b-catenin by 2.4. Reporter gene assay co-immunoprecipitation analysis. Flag-Glis2 and EGFP- Cells were transfected with the reporter plasmids pFR-LUC (Strat- agene), TOPFlash, FOPFlash, Super8XTOPFlash, Super8XFOP- b-catenin were transiently expressed in HCT116 cells and Flash, PCD1, pCMVbeta or pRL-SV40, and various Glis2 and Flag-Glis2 protein complexes immunoprecipitated using anti- b-catenin constructs as indicated. Transfection was performed with Flag-M2 agarose resin. As shown in Fig. 2A, Western blot Fugene 6 transfection reagent. phRL-SV40 served as internal control. analysis using an anti-GFP antibody identified EGFP-b-cate- Luciferase activity was measured 48 h later using the Dual-Luciferase Reporter Assay System from Promega. All transfections were nin as part of the immunoprecipitated protein complexes. performed in triplicate. Experiments were carried out at least twice. We next examined whether Glis2 was able to bind endogenous b-catenin in HCT116 cells expressing Flag-Glis2. As shown in Fig. 2B, endogenous b-catenin was co-immunoprecipitated 3. Results with anti-Flag-M2 affinity resin. Inversely, immunoprecipitation of protein complexes containing endogenous b-catenin 3.1. Identification of b-catenin as a Glis2-interacting protein by anti-b-catenin affinity resin co-immunoprecipitated Glis2 Previous studies have shown that Glis2 contains a transcrip- (Supplement Fig. 2). These results are in agreement with the tional activation as well as a repressor domain and that the data obtained by mammalian two-hybrid analysis and support transcriptional repression is mediated through interaction with the conclusion that Glis2 and b-catenin interact with each other proteins [4,7]. To identify additional proteins that mod- other. ulate or mediate Glis2 activity, we performed yeast two-hybrid To determine whether this interaction was specific for Glis2, cDNA screening using the amino-terminus (aa 1–196) and the we examined the interaction between b-catenin and another carboxyl-terminus (aa 319–522) of Glis2 as baits and two dif- member of the Glis subfamily. As shown in Fig. 2C, Glis2 ferent cDNA libraries as preys (see Supplements). Glis2 (319– but not Glis1 co-immunoprecipitated b-catenin. These data 522) turned out to be self-activating in yeast and therefore suggest that b-catenin specifically interacts with Glis2. could not be used in yeast-two hybrid analysis. However, yeast two-hybrid analysis with Glis2 (1–196) as bait yielded eight po- 3.2. In vitro interaction of Glis2 with b-catenin sitive clones encoding proteins with widely different functions To determine whether b-catenin physically interacted (Table 1). These included b-catenin, lysine-deficient protein ki- with Glis2, we performed in vitro pulldown analysis using nase 1 (WNK1), and two tetratricopeptide repeat domain con- GST-b-catenin and in vitro translated, [35S]-labeled Glis2.

Table 1 Putative Glis2-interacting proteins identified by yeast to hybrid analysis GenBank # Symbol Description Function AK035311 CTNNB Beta-catenin Cell adhesion and signal transduction NM_014309 RBM9 RNA binding motif protein 9, isoform 2 Negative regulator of transcription NM_015330 KIAA0376 Cytospin A Unknown NM_053193 CPSF1 Cleavage and polyadenylation specific factor 1 Nucleic acid binding NM_029522 GPSM2 G-protein signaling modulator 2 (AGS3-like) GTPase NM_133671 U2AF2 U2 small nuclear ribonucleoprotein auxiliary factor 2 RNA splicing NM_018979 WNK1 WNK lysine deficient protein kinase 1 Kinase, ion transport NM_020196 XAB2 XPA binding protein 2 DNA repair, transcription 860 Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864

GST-b-catenin was incubated with [35S]-labeled Glis2 and GST-b-catenin protein complexes isolated with glutathione– Sepharose beads. As shown in Fig. 3A, GST-b-catenin was able to pulldown radiolabeled Glis2 while GST (control) did not. Moreover, GST-b-catenin protein was unable to pulldown [35S]-labeled Glis1 in agreement with the results shown in Fig. 2C. These data support the conclusion that b-catenin and Glis2 physically interact with each other and demonstrate that this interaction is speciﬁc.

3.3. The first zinc finger motif of Glis2 is essential for its interaction with b-catenin To obtain greater insight into what region of Glis2 was involved in the interaction with b-catenin, we performed GST-pulldown analysis using GST-b-catenin and several [35S]-labeled Glis2 deletion mutants. Fig. 3B shows that GST-b-catenin was able to bind full-length Glis2 and Glis2 (170–520), but not Glis2 (1–169) or Glis2 (324–520). GST- Fig. 1. Identification of b-catenin as a Glis2-interacting protein by b-catenin also did not bind luciferase which was used as a mammalian two-hybrid analysis. HCT116 cells were co-transfected negative control. These results suggest that the zinc finger with (UAS)5-LUC, pRL-SV40, pM, pVP16, pM-Glis2, and increasing domain of Glis2, localized between amino acids 170 and 324, amounts of pVP16-b-catenin as indicated. After 48 h, cells were is important in the interaction with b-catenin. harvested and assayed for reporter activities. The relative LUC activity Co-immunoprecipitation analysis with V5-b-catenin and dif- was calculated and plotted. ferent Flag-Glis2 expression vectors supported the importance

Fig. 2. Glis2 and b-catenin are associated with the same protein complex. (A) HCT116 cells were transfected with p3xFlag-CMV-Glis2, p3xFlag- CMV, and pEGFP-wt-b-catenin as indicated. Forty-eight hours 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 immuno-precipitation (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 b-catenin. HCT116 cells were transfected with p3xFlag-CMV-Glis2 or p3xFlag-CMV. Forty-eight hours 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-b-catenin or anti-Flag antibodies. (C) The interaction of b-catenin is specific for Glis2. HCT116 cells were transfected with V5-b-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. Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864 861

Fig. 3. Glis2 interacts directly with b-catenin. (A) Interaction of GST and GST-b-catenin fusion proteins with [35S]-methionine-labeled Glis2 or Glis1 was examined as described in Section 2. First lane, 10% input; G, GST; C; GST-b-catenin. Arrow indicated Glis1 and arrow head indicated Glis2. (B) Glis2 interacts with b-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-b-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- Glis2ZF1mut containing the C175A mutation in ZF1 (ZF1mut). Proteins in the cellular extracts and immunoprecipitated proteins were examined by Western blot analysis using anti-Flag M2 or anti-V5 antibodies. of the zinc finger domain. Fig. 3C shows that b-catenin was full-length b-catenin, and the b-catenin mutants (1–423), able to immunoprecipitate full-length Glis2 and the mutants (132–423), and (423–781) were able to pulldown Glis2. In con- Glis2 (1–382), Glis2 (1–200), and Glis2 (170–377) but did not trast, control GST and the b-catenin mutant (1–131) did not immunoprecipitate Glis2 (1–143). These data suggest that the pulldown Glis2. These results indicate that the region of b- 1st zinc finger (ZF1) region of Glis2 (from aa 170 to 193) is catenin containing the armadillo repeats are important in its critical for its interaction with b-catenin. interaction with Glis2 but that the amino terminus is not re- To obtain further support for this conclusion, we ana- quired. lyzed the ability of b-catenin to interact with the mutant Glis2ZF1mut, which contains a C175A mutation in the first 3.5. Glis2 enhances the nuclear localization of b-catenin zinc finger that destroys the tetrahedral configuration in the We next examined the subcellular localization of Glis2 and zinc finger. Fig. 3D shows that V5-b-catenin did not co-immu- b-catenin in HEK293 cells by confocal microscopy. In 79% noprecipitate Glis2ZF1mut suggesting that the structural integ- of the cells expressing pEGFP-b-catenin only, b-catenin was rity of the first zinc finger of Glis2 is important for its found equally distributed between cytoplasm and nucleus, interaction with b-catenin. while in 21% of the cells b-catenin was confined largely to the nucleus (Fig. 5). However, when pEGFP-b-catenin and 3.4. The armadillo repeats of b-catenin are important for its Flag-Glis2 were co-expressed, the percentage of cells in which interaction with Glis2 b-catenin was restricted mainly to the nucleus, was greatly To determine which region of b-catenin is involved in its increased to about 90% (Fig. 5). Similar results were obtained interaction with Glis2, we performed GST-pulldown analysis with HCT116 cells (data not shown). These observations using GST-b-catenin and different mutant GST-b-catenin fu- suggest that Glis2 enhances the nuclear location of b-catenin. sion proteins and [35S]-labeled Glis2. As shown in Fig. 4, Under all conditions analyzed, Glis2 localized only to the 862 Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864

mutation in the 1st zinc finger motif, however, did not have any effect on the nuclear localization of Glis2. These observations are in agreement with our conclusion that the integrity of the 1st zinc finger domain of Glis2 is essential in its interaction with b-catenin.

3.6. Glis2 represses b-catenin/TCF-mediated transcriptional activation Previous studies have shown that Glis2 contains an activation and a repressor domain [4]. To determine whether Glis2 interfered with b-catenin-induced transcription, we analyzed the effect of Glis2 on TCF-mediated transcriptional activation using TOPFlash and FOPFlash TCF-reporters which contain multiple copies of an optimal TCF-binding element (TBE) and mutant TBE, respectively. As shown in Fig. 6A, pEGFP-DN90-b-catenin, a constitutively active form of b- catenin, induced reporter activity highly (6-fold) in HCT116 cells. This increase was greatly (67%) inhibited by co-expression of Glis2 suggesting that Glis2 inhibits TCF-mediated transcriptional activation (Fig. 6A). This was confirmed by reporter analysis using Super8XTOPFlash and Super8XFOP- Flash reporters which contain eight copies of TBE or mutant TBE, respectively [16]. The constitutively active b-catenin (S37C) caused a 7-fold increase in transcriptional activation, this activity was dramatically (70%) suppressed by Glis2 (Sup- Fig. 4. b-Catenin interacts with Glis2 through its specific regions. GST plement Fig. 3). No such effects were observed with the TCF and several GST-b-catenin fusion proteins were bound to glutathione- mutant reporter Super8XFOPFlash (Supplement Fig. 3). Glis2 35 Sepharose 4B beads and then incubated with [ S]-methionine-labeled repressed transcriptional activation by b-catenin in a dose Glis2. Bound radiolabeled proteins were visualized by autoradiogra- dependent manner (Fig. 6B). At high levels of Glis2 the repor- phy. The input of the various GST-b-catenin proteins is shown in the lower panel. Asterisks indicate the respective GST-b-catenin proteins. ter activity was below that of control HCT116 cells suggesting that Glis2 also inhibited activation by the constitutively active form of b-catenin present in these cells (compare bar 1 with bar 5, Fig. 6B). These data suggest that Glis2 functions as a negative modulator of b-catenin/TCF-mediated transcriptional activation.

3.7. Glis2 represses cyclin D1 promoter activity We next examined the repressor activity of Glis2 on the activation of the cyclin D1-promoter (PCD1), a well-studied target of the Wnt signaling pathway [17], in HCT116 cells. As shown in Fig. 6C, the constitutively active b-catenin (S37C) enhanced PCD1 reporter activity about 2.5-fold. Coexpression of Glis2 signiﬁcantly reduced cyclin D1-reporter activity in a dose- dependent manner (Fig. 6C). We next examined the eﬀect of b-catenin and Glis2 on the expression of endogenous cyclin D1 in HCT116 cells. The level of cyclin D1 protein was enhanced by b-catenin (Fig. 6D). Glis2 overexpression diminished the b-catenin-induced increase in cyclin D1. These results support the concept that Glis2 functions as a repressor of the activation of target genes in the Wnt/b-catenin signaling pathway.

Fig. 5. Glis2 enhances nuclear localization of b-catenin. NEH293 cells were transiently transfected with p3xFlag-CMV-Glis2, p3xFlag-CMV- 4. Discussion Glis2ZF1mut, and/or pEGFP-b-catenin. The percentage of cells in which the localization of Glis2 or b-catenin was restricted to the To obtain greater insights into the mechanisms by which nucleus, was calculated and plotted. The number of cells counted is Glis2 regulates transcription, we performed yeast two-hybrid indicated above each bar. screening using the amino-terminus of Glis2 as bait. This analysis identiﬁed several putative Glis2-interacting proteins, including WNK1, XAB1, GPSM2, and b-catenin. XAB1 and nucleus. In contrast to Glis2, Glis2ZF1mut did not change the GPSM2 contain a tetratricopeptide repeat domain which are nuclear translocation of b-catenin signiﬁcantly (Fig. 5). The thought to mediate protein–protein interactions in a variety Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864 863

Fig. 6. Glis2 represses TCF-mediated transcriptional activity and the induction of cyclin D1 by b-catenin. (A) HCT116 cells were cotransfected with TOPFlash and FOPFlash reporters, pCMVbeta, b-catenin (DN90), and p3xFlag-CMV-Glis2 as indicated. Forty-eight hours after transfection, cells were assayed for reporter activities as described in Section 2. (B) HCT116 cells were cotransfected with Super8XTOPFlash and Super8XFOPFlash reporters, pRL-SV40, pCMV-V5-b-catenin (b-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-b-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-b-catenin and p3xFlag-CMV-Glis2 (1.0 and 1.5 lg) as indicated. Thirty-six hours later, cellular proteins were analyzed by Western blot analysis using antibodies against V5, Flag, b-tubulin, and cyclin D1. The lower molecular weight band (arrow head) in the Flag-Glis2 Western blot represents a proteolytically processed form of Glis2. of biological systems [18,19]. b-Catenin is a versatile protein region contains the first zinc finger motif of Glis2. The critical with a function in cell adhesion and in the canonical Wnt sig- role of the first zinc finger motif was confirmed by the observa- naling pathway [8–10]. Binding of Wnt to receptors of the Friz- tion that a point mutation in the first zinc finger motif, that zled family results in the translocation of b-catenin to the destroys its tetrahedral configuration, abolished the interac- nucleus where it interacts with TCF family members replacing tion. b-Catenin contains several functional domains, the amino co-repressors and promoting the recruitment of co-activators terminus interacts with GSK3b and casein kinase Ia (CK1) and activation of transcription of target genes, including cyclin binding sites while its 12 armadillo repeats provides an inter- D1. face for TCF/LEFs, the co-activator CBP, and several other Because Glis proteins are closely related to members of the proteins [8–10]. Our results show that the amino-terminus of Gli subfamily [1,2,4,5] and because of reported interactions b-catenin is not essential for the interaction with Glis2 but that between the hedgehog/Gli and Wnt/b-catenin signaling path- its armadillo repeat domain is required. The interaction of ways [14,15] and the fact that two independent clones of b- b-catenin with TCF members, axin, cadherins, and the perox- catenin were isolated by yeast two-hybrid analysis, we decided isome proliferator-activated receptor c (PPARc) has been to focus on the interaction between b-catenin and Glis2. Mam- shown to involve the central region of the armadillo repeats malian two-hybrid and co-immunoprecipitation analysis con- while interaction with the androgen receptor involves the first firmed that b-catenin interacts with Glis2; this was further six armadillo repeats [13]. Each armadillo repeat consists of supported by GST-pulldown analysis. We demonstrated by three a-helices and all 12 repeats form a superhelical structure deletion analysis that the region between aa 170 and 193 is with a long positively charged groove [20]. Interestingly, the important for the interaction of Glis2 with b-catenin. This second loop in zinc finger 1 of Glis2 consists of a negatively 864 Y.-S. Kim et al. / FEBS Letters 581 (2007) 858–864 charged a-helix containing 3 Glu/Asp residues that could facil- novel Gli-related, Kruppel-like zinc finger protein containing itate the interaction of Glis2 with the armadillo repeats. transactivation and repressor functions. J. Biol. Chem. 277, Previous studies have demonstrated that Glis2 can function 30901–30913. [2] Kim, Y.S., Nakanishi, G., Lewandoski, M. and Jetten, A.M. as a repressor of gene transcription and recruit the co-repres- (2003) GLIS3, a novel member of the GLIS subfamily of sors CtBP1 and HDAC3 [7]. Our results indicate that Glis2 Kruppel-like zinc finger proteins with repressor and activation can inhibit b-catenin/TCF-mediated transcriptional activation functions. Nucleic Acids Res. 31, 5513–5525. of TOPFlash. In addition, expression of Glis2 represses the [3] Lamar, E., Kintner, C. and Goulding, M. (2001) Identification of NKL, a novel Gli-Kruppel zinc-finger protein that promotes promoter activity of the human cyclin D1 gene, a natural neuronal differentiation. Development 128, 1335–1346. Wnt target, and inhibits the induction of cyclin D1 expression [4] Zhang, F., Nakanishi, G., Kurebayashi, S., Yoshino, K., Peran- by b-catenin. These observations strongly suggest that Glis2 toni, A., Kim, Y.S. and Jetten, A.M. (2002) Characterization of may be a repressor of Wnt/b-catenin signaling. A number of Glis2, a novel gene encoding a Gli-related, Kruppel-like tran- other transcriptional factors, including several nuclear recep- scription factor with transactivation and repressor functions. Roles in kidney development and neurogenesis. J. Biol. Chem. tors, have been shown to be potent repressors of b-catenin/ 277, 10139–10149. TCF signaling [13]. This negative regulation can involve differ- [5] Kasper, M., Regl, G., Frischauf, A.M. and Aberger, F. (2006) ent mechanisms, including competition for a limited pool of b- GLI transcription factors: mediators of oncogenic Hedgehog catenin, enhanced degradation of b-catenin, competition for signalling. Eur. J. Cancer 42, 437–445. [6] Kinzler, K.W. and Vogelstein, B. (1990) The GLI gene encodes a co-factors, or recruitment of co-repressors to TCF/b-catenin nuclear protein which binds specific sequences in the human complexes. Since Glis2 acts as a repressor and associates with genome. Mol. Cell. Biol. 10, 634–642. CtBP1 and HDAC3, it may be unlikely that it competes with [7] Kim, S.C., Kim, Y.S. and Jetten, A.M. (2005) Kruppel-like zinc b-catenin/TCF for the binding of co-activators. The nuclear finger protein Gli-similar 2 (Glis2) represses transcription through proteins Pontin52 and Reptin52 have been reported to interact interaction with C-terminal binding protein 1 (CtBP1). Nucleic Acids Res. 33, 6805–6815. with the armadillo repeats of b-catenin and to form a multi- [8] Bienz, M. and Clevers, H. (2003) Armadillo/beta-catenin signals protein complex that includes TCF and TATA-box binding in the nucleus – proof beyond a reasonable doubt? Nat. Cell Biol. protein (TBP) [12]. Glis2 may be part of a similar multiprotein 5, 179–182. transcriptional complex. Elucidating the mechanisms by which [9] Sharpe, C., Lawrence, N. and Martinez Arias, A. (2001) Wnt signalling: a theme with nuclear variations. Bioessays 23, 311–318. Glis2 modulates transcription requires further study. 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