Glis3 is associated with primary cilia and Wwtr1/TAZ and implicated in polycystic kidney disease

MOLECULAR AND CELLULAR BIOLOGY, Mar. 2009, p. 2556–2569 Vol. 29, No. 100270-7306/09/$08.00�0 doi:10.1128/MCB.01620-08

Glis3 Is Associated with Primary Cilia and Wwtr1/TAZ andImplicated in Polycystic Kidney Disease�‡

Hong Soon Kang,1† Ju Youn Beak,1† Yong-Sik Kim,1 Ronald Herbert,2 and Anton M. Jetten1*Laboratory of Respiratory Biology, Cell Biology Section, Division of Intramural Research,1 and Laboratory of Experimental Pathology,

National Institute of Environmental Health Sciences,2 National Institutes of Health,Research Triangle Park, North Carolina 27709

Received 16 October 2008/Returned for modification 19 November 2008/Accepted 26 February 2009

In this study, we describe the generation and partial characterization of Kruppel-like zinc finger proteinGlis3 mutant (Glis3zf/zf) mice. These mice display abnormalities very similar to those of patients withneonatal diabetes and hypothyroidism syndrome, including the development of diabetes and polycystickidney disease. We demonstrate that Glis3 localizes to the primary cilium, suggesting that Glis3 is partof a cilium-associated signaling pathway. Although Glis3zf/zf mice form normal primary cilia, renal cystscontain relatively fewer cells with a primary cilium. We further show that Glis3 interacts with thetranscriptional modulator Wwtr1/TAZ, which itself has been implicated in glomerulocystic kidney disease.Wwtr1 recognizes a P/LPXY motif in the C terminus of Glis3 and enhances Glis3-mediated transcriptionalactivation, indicating that Wwtr1 functions as a coactivator of Glis3. Mutations in the P/LPXY motifabrogate the interaction with Wwtr1 and the transcriptional activity of Glis3, indicating that this motif ispart of the transcription activation domain of Glis3. Our study demonstrates that dysfunction of Glis3leads to the development of cystic renal disease, suggesting that Glis3 plays a critical role in maintainingnormal renal functions. We propose that localization to the primary cilium and interaction with Wwtr1 arekey elements of the Glis3 signaling pathway.

Gli-similar 1 to 3 (Glis1-3) constitute a subfamily of Krup-pel-like zinc finger proteins (4, 25, 27, 28, 30, 39, 56, 57). Glisproteins contain a DNA binding domain consisting of fiveC2H2-type zinc finger motifs that share a high degree of ho-mology with members of the Gli and Zic subfamilies of tran-scription factors (1, 24). Glis proteins regulate gene transcrip-tion by interacting with a specific nucleotide sequence, referredto as the Glis-DNA binding site (Glis-BS), in the promoterregion of target genes (3, 4). Glis1-3 proteins are expressed ina spatial and temporal manner during embryonic development,suggesting that they regulate specific physiological processes(25, 27, 28, 30, 39, 56). Loss of Glis2 function in mice andmutations in GLIS2 have been associated with nephronoph-thisis (2, 26), while genetic alterations in the GLIS3 gene havebeen linked to a syndrome characterized by neonatal diabetesand congenital hypothyroidism (NDH) (45, 47).

To obtain greater insights into the physiological functions ofGlis3 and its role in disease, we generated Glis3 mutant mice(Glis3zf/zf) in which the fifth zinc finger (ZF5) is deleted. ZF5is critical for the binding of Glis3 to Glis-BS and therefore forits transcriptional activity (3). We show that Glis3 mutant miceexhibit abnormalities very similar to those displayed by NDH1patients (45, 47), including a greatly reduced life span anddevelopment of polycystic kidneys and neonatal diabetes.

These similarities suggest that Glis3zf/zf mutant mice providean excellent model to study this syndrome.

This study focuses on the cystic renal phenotype of Glis3zf/zf

mutant mice. Cystic renal disease represents a heterogeneousgroup of genetic disorders, characterized by the developmentof multiple cystic lesions that could involve any segment of thenephron (36, 49). Autosomal dominant polycystic kidney dis-ease (PKD), autosomal recessive PKD, and nephronophthisisare the most studied variants of cystic renal disease. Interest-ingly, a large number of genes implicated in cystic renal diseaseencode proteins that are either localized to the primary ciliumor are part of a signaling pathway associated with ciliary func-tion (7, 12, 17, 36, 49, 50, 52, 54). These findings led to thehypothesis that dysfunction of the primary cilium and defectsin cilium-associated signal transduction pathways are key fac-tors in the etiology of cystic renal disease. Although the precisemolecular mechanisms responsible for cyst development haveyet to be established, it is thought that changes in cell-matrixand cell-cell interactions, Ca2� signaling, cell proliferation anddifferentiation, apoptosis, and cell polarity play critical roles inthis process (11, 29, 46).

In this study, we identify two key elements in the Glis3signaling pathway that are relevant to the development ofcystic kidney disease. We demonstrate that Glis3 is associatedwith the primary cilium, suggesting that activation of Glis3involves a primary cilium-associated signal pathway. In addi-tion, we show that Wwtr1, a WW domain-containing protein(also named TAZ) that functions as a modulator of severaltranscription factors (9, 19, 37, 51), interacts with and functionsas a coactivator of Glis3. Interestingly, Wwtr1 null mice them-selves have been reported to develop cystic renal disease thatresembles that with Glis3 (20, 34, 48). Our results indicate that

* Corresponding author. Mailing address: LRB, Cell Biology Sec-tion, Division of Intramural Research, National Institutes of Health,Research Triangle Park, NC 27709. Phone: (919) 541-2768. Fax: (919)541-4133. E-mail: [email protected].

‡ Supplemental material for this article may be found at http://mcb.asm.org/.

† These authors contributed equally to this study.� Published ahead of print on 9 March 2009.

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Glis3 and Wwtr1 are part of overlapping transcription regula-tory networks that play a critical role in the maintenance ofnormal renal architecture and function.

MATERIALS AND METHODS

Generation of Glis3zf/zf mice. Glis3 genomic flanking regions were generatedby PCR amplification using 129/Sv genomic DNA as a template. A 4.7-kb XbaI/ClaI fragment of intron 3 and a 3.0-kb BamHI/NotI fragment of intron 4 wereinserted into the NheI/ClaI and BamHI/NotI sites of pOSdupdel. The resultingpOSdupdel-Glis3 plasmid DNA was linearized by NotI and electroporated into129/Sv embryonic stem (ES) cells (Randy Thresher, UNC Transgenic Facility).Gene-targeted ES cells were microinjected into blastocysts from C57BL/6 micethat were then returned to pseudo-pregnant B6D2 mice. Chimeric mice werecrossed with C57BL/6 mice to identify transmitting chimeras and to obtain miceheterozygous for the mutant allele. Heterozygous mice were intercrossed toobtain animals homozygous for the mutant allele and to obtain wild-type (WT)littermate controls. All animal studies followed guidelines outlined in the NIHGuide for the Care and Use of Laboratory Animals, and protocols were ap-proved by the Institutional Animal Care and Use Committee at the NIEHSand UNC.

Genotyping and Southern blot analysis. DNA (8 �g) prepared from ES cellsand tail biopsy specimens was digested with KpnI or XbaI and separated byelectrophoresis in a 1% agarose gel containing 1� Tris-acetate-EDTA buffer.After transfer to an Immobilon-Ny� membrane (Millipore), DNA was hybrid-ized to [�-32P]dCTP-labeled 5�-end or 3�-end probes, respectively. Routine geno-typing was carried out with the following primers: Glis3-F, 5�-AGCTAGTGGCTTTCGCCAACA-3�; Glis3-R, 5�-GAACAAGATAGAATCATGGTATATCC-3�; and Neo-pro, 5�-ACGCGTCACCTTAATATGCG-3�.

Histology and immunohistochemical staining. Kidneys were fixed in 10%neutral buffered formalin for 24 h, subsequently transferred to 70% ethanol,processed, embedded in paraffin, sectioned at 5 �m, and stained with hematox-ylin/eosin. To identify proximal tubules, collecting ducts, distal tubules, or pri-mary cilium, frozen sections were stained with fluorescein isothiocyanate(FITC)-labeled Lotus tetragonolobus lectin (LTA), FITC-labeled Dolichos biflo-rus agglutinin (DBA) (Vector Laboratory, Burlingame, CA), calbindin-D-28K,or anti-acetylated �-tubulin antibodies (Sigma, St. Louis, MO), respectively. Toexamine cell polarity, sections were stained with antibodies against epidermalgrowth factor (EGF) receptor (Sigma), �-catenin (BD Bioscience), ZO-1(Zymed, San Francisco, CA), or Na-K-ATPase � (Santa Cruz Biotechnology).Alexa Fluor-488- or -594-conjugated antibodies (Molecular Probes, Eugene,OR) were used as secondary antibodies. Fluorescence was observed with a LeicaDMRBE microscope (Leica, Wetzlar, Germany). To analyze proliferating cellslining the glomeruli cyst, 50 mg/kg of bromodeoxyuridine (BrdU) was injectedinto postnatal day 3 (PND3) pups. After 2 h, kidneys were collected and fixed.Sections were selected randomly from three mice for each group and stainedwith a BrdU antibody. The percentage of BrdU-positive cells was calculated fromsix to seven glomeruli in each section and plotted.

In situ hybridization. In situ hybridization on frozen tissue sections was carriedout by Phylogeny (Columbus, OH) using a 35S-UTP-labeled, Glis3-specific probeas described previously (28).

Glis3 knockdown and QRT-PCR analyses. Total RNA from cells or tissueswas isolated using mini or midi RNA isolation kits (Qiagen, Valencia, CA)according to the manufacturer’s instructions (23). In Glis3 knockdown experi-ments, TKPTS cells were transfected with Glis3 small interfering RNAs(siRNAs) from Ambion (Austin, TX) using Lipofectamine 2000 (Invitrogen,Carlsbad, CA), and 48 h later RNA was isolated. Equal amounts of total RNAwere reverse transcribed using a high-capacity cDNA archive kit (Applied Bio-systems) and then examined by real-time quantitative PCR (QRT-PCR) analysis.The PCRs were carried out in triplicate in a 7300 QRT-PCR system (AppliedBiosystems, Foster City, CA) as previously described (4). All results were nor-malized to the 18S transcript. Primers were designed using Primer Express 2.0software and synthesized at Sigma/Genosys (St. Louis, MO). Primers and probesare shown in Table S1 in the supplemental material.

Plasmids. The reporter plasmid pFR-Luc (Stratagene), p-TAL-Luc-(Glis-BS)6, and several p3�FLAG-CMV-Glis3 expression vectors were described pre-viously (3, 4). p3�FLAG-CMV-Glis1 and p3�FLAG-CMV-Glis2 were de-scribed previously (25, 27). p3�FLAG-CMV-Glis3M4 containing the pointmutations P843A and Y845A was generated using a QuikChange site-directedmutagenesis kit (Stratagene, La Jolla, CA). pcDNA3.1-Wwtr1(TAZ) was kindlyprovided by Ikramuddin Aukhil (University of Florida, Gainesville, FL). pCMV-myc-Wwtr1 was generated by PCR amplification using pcDNA3.1-Wwtr1 as a

template, and the PCR product was inserted into the EcoRI and KpnI sites ofpCMV-myc. The sequence of each insert was verified by restriction enzymemapping and DNA sequencing.

Confocal microscopy. The subcellular localization of Glis3 and Wwtr1 wasexamined by confocal microscopy largely as described previously (3). COS-1 cellswere plated and transfected with pEGFP-Glis3, pEGFP-Glis3M4 or pEGFP-Glis3�ZF5 with or without p3�FLAG-CMV-Wwtr1. After 30 h of incubation,cells were fixed and stained with anti-FLAG M2 mouse monoclonal antibody(Sigma) and subsequently with a goat anti-mouse Alexa Fluor-594 antibody(Molecular Probes). Fluorescence was observed in a LSM 510 UV Zeiss confocalmicroscope.

Cell culture and transactivation assay. The mouse kidney proximal tubule cellline TKPTS was kindly provided by Elsa Bello-Reuss (Texas Tech University,Lubbock, TX) and cultured in Dulbecco’s minimal essential medium/F-12 me-dium supplemented with 7% fetal bovine serum and 50 �g/ml insulin. To analyzeGlis3 transcriptional activity, HEK293T or COS-1 cells (ATCC) were cotrans-fected with p-TAL-Luc-(Glis-BS)6, p3�FLAG-CMV-Glis3, p3�FLAG-CMV-Glis3M4, pCMV-myc-Wwtr1, and pCMV� using Fugene 6 transfection reagent(Roche) as described previously (3). For mammalian two-hybrid analysis, cellswere transfected with the reporter plasmid pFR-Luc, pCMV�, pM-Wwtr1,pVP16-Glis3, or pVP16-Glis3M4 as indicated. Cells were incubated for 30 h andthen assayed for reporter activity. Luciferase and �-galactosidase activity wereassayed with a luciferase kit (Promega) or a luminescent �-galactosidase detec-tion kit (Clontech). Transfections were performed in triplicate, and each exper-iment was repeated at least twice.

Coimmunoprecipitation analysis. HEK293 cells were transiently transfectedwith pCMV-Myc-Wwtr1 and p3�FLAG-CMV-Glis3 expression vectors contain-ing Glis3, Glis3�N463, Glis3�C756, Glis3M4, or Glis3�ZF5. Forty-eight hours aftertransfection, cells were harvested and lysed in radioimmunoprecipitation assaybuffer (Upstate, Charlottesville, VA) containing protease inhibitor cocktails Iand II (Sigma) as described previously (25). Myc-Wwtr1 protein complexes werethen isolated using agarose A and Myc antibody (Invitrogen, Carlsbad, CA).Bound protein complexes were then solubilized in sample buffer and analyzed byWestern blot analysis using mouse anti-Flag M2 antibody (Sigma).

GST pull-down assay. Equal amounts of glutathione S-transferase (GST)-Wwtr1 or GST protein were incubated with glutathione-Sepharose 4B beads andthen washed in phosphate-buffered saline. [35S]methionine-labeled Glis3 andGlis3M4 were generated using a TNT quick coupled transcription/translationsystem (Promega). The GST- and GST-Wwtr1-bound beads were then incubatedwith [35S]methionine-labeled Glis3 in 0.5 ml of binding buffer (20 mM Tris-HCl,pH 7.6, 100 mM KCl, 0.05% Nonidet P-40, 0.1 mM EDTA, 10% glycerol, 0.2%Tween 20, 1 mM phenylmethylsulfonyl fluoride). After 1 h of incubation at roomtemperature, beads were washed five times in binding buffer and boiled in 15 �l2� sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer.Proteins were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and visualized by autoradiography.

RESULTS

Generation of Glis3 mutant mice. To obtain greater insightsinto the physiological functions of Glis3 and its role in NDH,we generated mice, referred to as Glis3zf/zf, in which the Glis3gene was disrupted. We selected a strategy that would result inthe deletion of exon 4, which encodes ZF5, and parts of introns3 and 4 (Fig. 1A; see Fig. S1 in the supplemental material).Deletion of ZF5 is predicted to disrupt the DNA bindingability and, therefore, the transcriptional activity of Glis3 (3).The latter was confirmed by experiments showing that, in con-trast to WT Glis3, Glis3 lacking ZF5 (Glis3�ZF5) was unable toinduce Glis-BS-dependent transcriptional activation of a lucif-erase reporter (Fig. 1B). However, in transiently transfectedcells, deletion of ZF5 did not affect the ability of Glis3 totranslocate to the nucleus (Fig. 1C and D). The loss of tran-scriptional activity of Glis3�ZF5 is consistent with our recentobservations showing that ZF5 is critical for the interaction ofGlis3 with Glis-BS (3).

Glis3zf/zf mice die shortly after birth. Glis3�/zf mating pairsproduced offspring with all three genotypes with the expected

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FIG. 1. Targeting the Glis3 locus. (A) Schematic view of the mouse Glis3 locus, the targeting vector pOSdupdel-Glis3, and the recombinationat the Glis3 locus. In the targeted locus, the region encoding the entire exon 4, ZF5, and parts of introns 3 and 4 are deleted. (B) Mutant Glis3�ZF5,lacking ZF5, is unable to induce Glis-BS-mediated transcription. COS-1 or C3H10T1/2 cells were cotransfected with the (Glis-BS)6-LUC reporterplasmid, pCMV�, and different concentrations of p3�FLAG-CMV-Glis3 or p3�FLAG-CMV-Glis3�ZF5 expression plasmid as indicated. After48 h of incubation, cells were collected and assayed for reporter activities, and the relative LUC reporter activities (RLA) were calculated andplotted. (C, D) COS-1 cells were transfected with p3�FLAG-CMV-Glis3 or p3�FLAG-CMV-Glis3�ZF5 expression plasmid, and 48 h later, thesubcellular localization of Glis3 was examined by confocal microscopy as described in Materials and Methods. (E) Glis3zf/zf mice have a very shortlife span. The survival of WT and Glis3zf/zf pups was monitored over time. The percentage of surviving mice was calculated and plotted againstthe age of the mice. (F) Glis3zf/zf mice have a normal appearance and show milk bands (arrows).

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Mendelian distribution, suggesting that deletion of exon 4 didnot cause embryonic lethality (data not shown). HomozygousGlis3zf/zf pups were smaller (about 10% reduction in weight;see Fig. S1D in the supplemental material) and displayed adramatically shortened life span (Fig. 1E). At PND3, onlyabout 50% of the Glis3zf/zf pups survived, while none of themlived longer than 11 days. The general appearance of PND3Glis3zf/zf mice was normal and milk bands were observed, sug-gesting that mice did not appear to exhibit any severe abnor-malities that interfered with their ability to drink or swallow(Fig. 1F). Heterozygous Glis3�/zf mice displayed no obviousphenotype and were indistinguishable from WT mice, asjudged by general appearance and life span.

Glis3zf/zf mice develop polycystic kidneys. Histopathologicalanalysis of PND3 Glis3zf/zf mice showed that the corticomed-ullary junction is consistently abnormal in Glis3zf/zf kidneys(Fig. 2; also see Fig. S2 in the supplemental material). Inaddition to there being multiple, fluid-filled cysts in both thecortex and medulla, the corticomedullary junction appearedless complex and showed an increased abundance of mesen-chymal cells. At PND3, all Glis3zf/zf mice developed cystictubules and glomerular cysts; however, not all tubules andglomeruli were cystic. Male and female Glis3zf/zf mice wereabout equally affected, while no renal cysts were observed inGlis3zf/� mice, suggesting the recessive nature of this mutation.The severity of the cystic phenotype was variable, and the sizeand number of cysts increased with age (Fig. 2B, D, and F).Abnormalities in Glis3zf/zf kidneys were first observed aroundembryonic day 14.5 (E14.5) to E15.5; dilations of Bowman’sspaces were the most obvious (Fig. 2). Cystic glomeruli couldoften be identified by the presence of glomerular tufts. Thesecysts became increasingly larger in diameter at PND0 andPND3 (Fig. 2). At PND3, Glis3zf/zf mice did not develop pro-teinurea (see Fig. S2C in the supplemental material) and nochange was observed in urinal pH or ketone levels (data notshown). In addition to polycystic kidneys, Glis3zf/zf mice devel-oped overt neonatal diabetes, evidenced by hyperglycemia andhypoinsulinemia caused by a deficiency in the generation ofpancreatic �-cells (data not shown; H. S. Kang, Y.-S. Kim, J. Y.Beak, G. Kilic, B. Sosa-Pineda, J. Jensen, J. Foley, and A. M.Jetten, submitted for publication).

We reported previously that during early rat kidney devel-opment, Glis3 is highly expressed in the ureteric bud (28). Insitu hybridization analysis revealed that at PND10, Glis3 ismost highly expressed in kidney (see Fig. S3A to C in thesupplemental material), consistent with the reported Northernblot analysis of Glis3 expression (28). Glis3 expression wasdetected in the epithelia of the collecting ducts and tubules andin the parietal layer of the glomerulus (see Fig. S3 in thesupplemental material). These observations suggest that thecystic phenotype in Glis3zf/zf mice is related directly to alter-ations in parietal cells and epithelial cells lining the tubules andcollecting ducts. Thus, expression of Glis3 overlaps with that ofmany other genes that have been implicated in renal cysticdisease, including Pkd1 and -2, Pkhd1, Wwtr1, and Hnf1� (15,20, 21, 34, 36, 48–50).

Cysts in PND3 Glis3zf/zf mice originate from glomeruli, tu-bules, and collecting ducts. To determine the origins of thecysts, kidneys were stained with LTA, calbindin-D-28K anti-body, and DBA, which specifically label the proximal tubules,

distal tubules, and the collecting ducts, respectively. At PND3,most of the larger cysts originated from glomeruli, while manyproximal and distal tubules and collecting ducts in kidneys ofGlis3zf/zf mice were dilated (Fig. 2J to O). Of the total cysticvolume, about 65% originated from glomeruli, 4% from col-lecting ducts, 4% from proximal tubules, and 11% from distaltubules. The origin of the remaining 16% could not be deter-mined.

Increased cell proliferation has been proposed to be in-volved in cyst formation. We therefore analyzed proliferationin kidneys from WT and Glis3zf/zf mice by BrdU labeling.Because the largest cysts originated from glomeruli, we com-pared the percentage of BrdU-positive cells lining Bowman’sspaces with that of glomerulocysts in Glis3zf/zf mice. The per-centage of BrdU-positive cells was significantly higher in theepithelium lining glomerulocysts than that of normal Bow-man’s spaces (Fig. 2I). Moreover, immunofluorescent stainingshowed that cells in normal tubules contained significantlyhigher levels of the cyclin-dependent kinase inhibitor cdkn1a(p21cip1), which is known to be associated with growth arrest/inhibition, than in cystic tubules (see Fig. S4A in the supple-mental material). Together these data suggest that Glis3 dys-function leads to increased cell proliferation of cells liningBowman’s spaces and renal tubules. Whether the observedincrease in proliferation is a consequence or cause of cystformation needs to be determined further.

Glis3 localizes to primary cilium. A large number of genesimplicated in cystic renal disease encode proteins that eitherlocalize to the primary cilium or are part of a signaling pathwaylinked to ciliary function (7, 12, 49, 54). This raised the ques-tion of whether Glis3 is also associated with the primary ciliumand whether there is a link between the cystic renal phenotypeobserved in Glis3zf/zf mice and the primary cilium. This ideawas strengthened by recent observations demonstrating thatanother member of the Glis subfamily, Glis2, and the closelyrelated Gli proteins have been reported to be associated withthe primary cilium (2, 14, 33). Primary cilia were visualized byimmunofluorescent staining with an anti-acetylated �-tubulinantibody. Immunofluorescent microscopic analysis showedthat Glis3 localized to the primary cilia of tubule epithelialcells in sections from mouse kidney, as indicated by its colo-calization with acetylated �-tubulin (Fig. 3A to C). This wasfurther supported by experiments examining the subcellularlocalization of Glis3 in cultured mouse kidney proximal tubuleTKPTS cells transiently expressing enhanced green fluorescentprotein (EGFP)-Glis3. To promote formation of primary ciliain these cells, cultures were maintained at confluence and inlow serum for several days. Confocal microscopy showed thatEGFP-Glis3 is localized to the primary cilium in addition tothe nucleus (Fig. 3D to F; see Fig. S5A to C in the supplemen-tal material). In the primary cilium, the fluorescent staining ofEGFP-Glis3 overlapped that of acetylated �-tubulin, and inseveral instances, Glis3 appeared to localize preferentially tothe tip of the primary cilium (Fig. 3D to F). These observationssuggest that Glis3 may be part of a primary cilium-associatedsignaling pathway.

Glis3 mutant mice are able to form primary cilia. Cysticrenal disease has been linked to various abnormalities in theformation of the primary cilium or ciliary function (29, 46, 54).Therefore, we examined whether the formation of primary



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cilia was affected in Glis3zf/zf mice. Most epithelial cells liningthe tubules and collecting ducts of kidneys of PND3 WT micecontained a primary cilium as indicated by staining for acety-lated �-tubulin (Fig. 3G). In Glis3zf/zf mice, the percentage ofcells with cilium lining normal renal tubules was not signifi-

cantly altered compared to that in WT mice; however, thispercentage was greatly decreased in dilated tubules and furtherreduced in cysts (Fig. 3H to J).

Next, we examined the epithelial lining of renal cysts inGlis3zf/zf mice by scanning electron microscopy. This analysis

FIG. 2. Glis3zf/zf mutant mice develop polycystic kidneys. Sections of kidneys from E15.5 (A, B), PND0 (C, D), and PND3 (E, F) WT (�/�)and Glis3zf/zf (zf/zf) mice were stained with hematoxylin/eosin. Dilation of glomeruli was observed in kidneys of E15.5 Glis3zf/zf mice (B). Dilationof tubules and glomeruli becomes more severe with age (B, D, F). Glomerular cysts (indicated by arrowheads) are the most prominent. (G, H)High magnification of a normal glomerulus and glomerular cyst of WT and Glis3zf/zf PND3 kidneys, respectively. Scale bars, 300 �m (A-F) and25 �m (G, H). (I) Increase in the percentage of BrdU-positive cells lining glomerular cysts of Glis3zf/zf PND3 kidneys compared to that of parietalcells lining normal Bowman’s spaces. Random sections were selected from kidneys of three mice in each group, and the percentage ofBrdU-positive cells lining Bowman’s spaces and glomerulocysts was determined as described in Materials and Methods. (J to O) Dilation and cystformation in renal tubules and collecting ducts in kidneys of Glis3zf/zf mice. Kidneys of PND3 WT (�/�) and Glis3zf/zf (zf/zf) mice were stainedwith LTA (J, M), calbindin-D-28K antibody (K, N), and DBA (L, O), markers for proximal tubules, distal tubules, and collecting ducts,respectively. Nuclei were stained with DAPI. Scale bars, 100 �m.



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confirmed that many cells in renal cysts contained a normalprimary cilium; however, a significant number of cells lacked aprimary cilium and a few contained a truncated primary cilium(Fig. 3K). These observations are consistent with our conclu-sion that Glis3 dysfunction leads to a significant reduction inrenal epithelial cells possessing a primary cilium. However,

many cells still contained what appeared to be a structurallynormal cilium. These results suggest that Glis3 is not essentialfor the formation of the primary cilium. Consistent with this isthe observation that TKPTS cells in which Glis3 expression wasdownregulated by Glis3 siRNAs are still able to form a primarycilium (see Fig. S5D in the supplemental material). The reduc-

FIG. 3. Glis3 protein localizes to the primary cilium. (A to C) Immunofluorescent staining of sections of WT kidney with antibodies againstacetylated �-tubulin (A) to detect primary cilia and Glis3 (green, B; merged, C). (D to F) Mouse kidney proximal tubule TKPTS cells transientlytransfected with pEGFP-Glis3 were maintained at confluence to induce the formation of primary cilia as described in Materials and Methods. Thesubcellular localization of EGFP-Glis3 (green) and acetylated �-tubulin was analyzed by confocal microscopy. Arrowheads indicate a primarycilium in which the patterns of fluorescent staining for EGFP-Glis3 and acetylated �-tubulin overlap; the insets show a higher magnification.Arrows indicate fluorescence staining for acetylated �-tubulin in Glis3-negative cells. (G to K) Glis3zf/zf mice are able to form normal primary cilia,but the percentage of ciliated cells lining dilated and cystic renal tubules and collecting ducts is reduced. (G to I) Kidney sections were stained withacetylated �-tubulin antibody (red) to detect primary cilia (arrows), BDA (green) to indicate collecting ducts, and DAPI (blue). Most cells liningcollecting ducts in WT kidneys contain a primary cilium (G); the relative number of cells with cilium was significantly reduced in dilated and cysticducts of Glis3zf/zf kidneys (H, I). Scale bars, 20 �m (G to I). (J) The percentage of cells with a primary cilium was reduced in renal tubules ofGlis3zf/zf kidneys. The percentage of cells with a primary cilium in normal renal tubules of WT kidneys and in dilated and cystic renal tubules ofGlis3zf/zf kidneys was calculated. Total numbers of cells counted are as follows: normal tubules in WT kidneys (n � 118); Glis3zf/zf kidneys withnormal tubules (n � 134), dilated tubules (n � 320), and cysts (n � 142). (K) Scanning electron microscopy of cells lining a renal cyst of a Glis3zf/zf

kidney. Many cells have a normal cilium (arrows), some cilia are disrupted (arrowheads), and some cells do not have a primary cilium.



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tion in cells with a primary cilium may be due to an indirectmechanism, such as increased proliferation or changes in cell-cell interactions.

Cell-cell interactions and gene expression in kidneys ofGlis3zf/zf mice. The development of renal cysts has been re-ported to be associated with changes in cell-cell and cell-matrixinteractions and cell polarity (36, 46, 49). We therefore com-pared the localizations and expression level of �-catenin, zonaoccludens 1 (ZO-1), and EGF receptor (EGFR), proteins thateither are part of junctions or function as markers of cellpolarity in the kidneys of WT and Glis3zf/zf mice. In normaltubules, �-catenin localized to the basolateral membrane (Fig.4A to C), but in regions of cystic distal tubules of Glis3zf/zf

mice, it was found to a significant degree in the cytoplasm aswell as in the apical membrane (Fig. 4D to F). ZO-1, which innormal distal tubules localized at tight junctions in the apicalmembrane (Fig. 4G to I), was highly localized in the basolat-eral membrane in Glis3zf/zf cystic tubules (Fig. 4J to L). Insome PKD models, including Kif3a mutant mice (32), alter-ations in cell polarity are associated with localization of theEGFR to the apical membrane instead of the basal membrane;however, in Glis3zf/zf mice, EGFR remained largely confinedto the basal membrane (Fig. 4M to R). Na�-K� ATPase �,which is normally restricted to the basal membrane, exhibiteda basolateral pattern of subcellular distribution in tubules ofGlis3zf/zf kidneys (see Fig. S4B in the supplemental material).These results suggest that Glis3 dysfunction results in changesin cell-cell interactions. This was supported by experimentsexamining the effect of Glis3 knockdown on the distributionpattern of EGFR, ZO-1, and E-cadherin in TKPTS cells. Allthree markers appeared absent or were weaker at the leadingedge of migrating control TKPTS cells than in TKPTS cells inwhich Glis3 was downregulated (see Fig. S6 in the supplemen-tal material). Interestingly, expression of polycystin-1 (PC-1,which is encoded by Pkd1) in MDCK cells has been reportedto induce a pattern of ZO-1 and E-cadherin distribution that isvery similar to that of control TKPTS cells expressing Glis3 (8).Thus, expression of Glis3 or Pkd1 has similar effects on thedistribution of several junctional proteins, including ZO-1 andE-cadherin.

Since Glis3 functions as a regulator of gene transcription, itis likely that the formation of cysts in Glis3zf/zf mice is causedby changes in the expression of specific Glis3 target genes. Wetherefore compared the expression levels of several genes pre-viously reported to be associated with cystic renal disease andciliary function in kidneys of WT and Glis3zf/zf mice. Theexpression of most of the genes examined, including Pkhd1,Nphp1-4, Kif3�, Umod1, Pkd2, Wwtr1, and Hnf1�, was notsignificantly altered in kidneys of Glis3zf/zf mice (Fig. 5A). Incontrast, the expression of Dctn5 and Pkd1 was decreased (by60 and 20%, respectively) in kidneys of Glis3zf/zf mice. Thedownregulation of Dctn5 and Pkd1 was confirmed in TKPTScells in which Glis3 expression was downregulated by Glis3-specific siRNA (Fig. 5B).

Glis3 interacts with the transcriptional modulator Wwtr1.The cystic phenotype of Glis3zf/zf mice exhibit a number ofsimilarities with cystic renal diseases in humans and in trans-genic mice caused by mutations in other genes, includingHNF1�, Wwtr1, and PKHD1 (15, 20, 21, 34, 35, 48). Thesimilarity between the renal phenotype of Glis3zf/zf and

FIG. 4. Changes in cell-cell interactions in cystic tubules ofGlis3zf/zf kidneys. The localization of �-catenin (A to F), ZO-1 (G toL), and EGFR (M to R) was examined in distal tubules from WT andGlis3zf/zf mice using specific antibodies and an Alexa Fluor-594-conju-gated secondary antibody (red). Distal tubules were identified by cal-bindin-D-28K antibody staining (green). Nuclei were stained withDAPI. Arrows indicate the normal distribution of the proteins, arrow-heads indicate an abnormal distribution. Scale bars, 25 �m.



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Wwtr1/ mice was particularly intriguing because cysts orig-inating from glomeruli are prominent in the kidneys of bothmutant mice. In addition to the apparent similarity in renalphenotype, Wwtr1 and Glis3 expression in the kidney overlapeach other, and both Wwtr1 and Glis3 have been reported tohave a stimulatory role in osteogenesis while inhibiting adipo-genesis (4, 19, 20, 28, 34). This comparison raised the questionof whether there was a functional link between the two pro-teins. This idea was strengthened by reports showing thatWwtr1 acts as a modulator of gene transcription by interactingwith several transcription factors (9, 18, 38, 42). These inter-actions are mediated through the WW domain of Wwtr1,which recognizes a P/LPXY motif (in which X is any aminoacid) in these transcription factors (22). We hypothesized thatWwtr1 might interact with Glis3 and enhance its transcrip-tional activity by acting as a coactivator of Glis3. This conceptwas supported by examination of the Glis3 sequence, whichidentified four potential P/LPXY motifs in Glis3, three at theN terminus before the zinc finger domain, and one at its Cterminus (Fig. 6A). To determine whether Wwtr1 functions asa transcriptional mediator of Glis3, we first examined the in-teraction between Glis3 and Wwtr1 by coimmunoprecipitationanalysis. These results showed that Glis3 was able to coimmu-noprecipitate Wwtr1, suggesting that Glis3 and Wwtr1 are partof the same protein complex (Fig. 6B). To establish which, if

any, of the putative P/LPXY motifs are involved in the inter-action of Glis3 with Wwtr1, we examined the effect of severaldeletion mutations within Glis3 on its interaction with Wwtr1.As shown in Fig. 6B, although deletion of the N terminus,including three of the four P/LPXY motifs of Glis3, had someeffect on the interaction with Wwtr1, deletion of the C termi-nus, including the fourth motif, abolished this interaction al-most completely, indicating a major role for the C terminus inthe interaction of Glis3 with Wwtr1. Moreover, these observa-tions suggested that of the four motifs, the fourth P/LPXYmotif at the C terminus is the most important for this interac-tion. This was supported by data showing that mutant Glis3M4,which contains the P841A, Y843A double point mutation in theC-terminal P/LPXY motif, was unable to interact effectivelywith Wwtr1. Deletion of the ZF5 motif, which matches themutation in Glis3zf/zf mice, did not greatly affect the interactionof Glis3 with Wwtr1 (Fig. 6B), indicating that Glis3�ZF5 caninteract with Wwtr1.

The interaction between Glis3 and Wwtr1 was further inves-tigated by mammalian two-hybrid analysis in 3T3-L1 cellstransfected with a (UAS)5-Luc reporter, pM-Wwtr1, andpVP16-Glis3 plasmid DNA. As shown in Fig. 6C, cotransfec-tion of pM-Wwtr1 with increasing amounts of pVP16-Glis3significantly enhanced the transcriptional activation of the Lucreporter in a concentration-dependent manner, whereas co-

FIG. 5. (A) Comparison of the expression levels of several genes previously implicated in PKD between kidneys from WT and Glis3zf/zf mice.RNA was isolated from kidneys of PND3 WT (n � 6) and Glis3zf/zf (n � 6) mice and then examined by QRT-PCR analysis. The expression levelof each gene was normalized to 18S rRNA. *, indicates P of 0.05 (compared to WT). (B) Effect of Glis3 downregulation on the expression ofPkd1 and Dctn5 in TKPTS cells. TKPTS cells were transfected with scrambled siRNA (“C”) or two different Glis3 siRNAs (si#1 or si#2), and48 h later, they were analyzed for the expression of Glis3, Pkd1, and Dctn5 mRNA by QRT-PCR. *, indicates P of 0.01 (compared to “C”).



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transfection with pVP16-Glis3M4 had little effect. These datasupport the conclusion that the interaction of Glis3 and Wwtr1is dependent on the C-terminal P/LPXY motif.

Subsequently, we examined the interaction of Glis3 withWwtr1 by in vitro pull-down analysis using purified GST-Wwtr1 fusion protein and 35S-labeled Glis3. This analysis

showed significant binding of Glis3 to Wwtr1, while GST alonedid not bind Glis3 (Fig. 6D). To examine whether the C-terminal P/LPXY motif of Glis3 was required for this interac-tion, GST pull-down analysis was also performed with theGlis3M4 mutant. As shown in Fig. 6D, Glis3M4 did not inter-act efficiently with GST-Wwtr1. These results are in agreement

FIG. 6. Glis3 interacts with the transcriptional modulator Wwtr1. (A) Glis3 contains four putative P/LPXY Wwtr1 interaction motifs. Thesequence of the four P/LPXY motifs of Glis3 are shown, as well as a schematic view of the location of these sites and the different Glis3 mutantsused in panel B. ZFD, zinc finger domain. (B) Human kidney HEK293T cells were transfected with p3�FLAG-CMV expressing Glis3, Glis3�N,Glis3�C, Glis3M4, Glis3�ZF5 (containing a mutation in the fourth Wwtr1 interaction motif), or Glis3�ZF5 with or without pCMV-myc-Wwtr1, asindicated. After 24 h of incubation, myc-Wwtr1 protein complexes were isolated with anti-myc agarose resin and examined by Western blot analysiswith antibodies against FLAG and myc. (C) Mammalian two-hybrid analysis. Cells were cotransfected with (UAS)5-Luc, pCMV�, pM-Empty orpM-Wwtr1, VP16-Empty, VP16-Glis3, or VP16-Glis3M4, as indicated. After 30 h of incubation, cells were collected and reporter activity wasdetermined. Luc activity was normalized against �-galactosidase. (D) In vitro pull-down of Glis3 by GST-Wwtr1. GST and GST-Wwtr1 (Wwtr1)fusion proteins were bound to glutathione-Sepharose 4B beads and then incubated with [35S]methionine-labeled Glis3 or Glis3M4. After 1 h ofincubation, beads were washed extensively and bound proteins were solubilized. Radiolabeled proteins were visualized by autoradiography. IPindicates 10% input of Glis3WT or Glis3M4. (E) Glis3 promotes the nuclear localization of Wwtr1. COS-1 cells were transfected withp3�FLAG-CMV-Wwtr1, pEGFP-Glis3WT, pEGFP-Glis3M4, or pEGFP-Glis3�ZF5 alone or cotransfected with p3�FLAG-CMV-Wwtr1. Thesubcellular localization of the fusion proteins was examined 30 h later by confocal microscopy as described in Materials and Methods. (F) Thepercentage of cells described for panel A, in which Wwtr1 was localized predominantly to the cytoplasm or nucleus or distributed about equallybetween nucleus and cytoplasm, was calculated and plotted.



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with the conclusion that the fourth P/LPXY motif plays amajor role in the interaction between Glis3 and Wwtr1.

Next, we examined the subcellular localization of Glis3 andWwtr1 in COS-1 cells. In virtually all cells (95%) transfected withpEGFP-Glis3 or pEGFP-Glis3M4 only, Glis3 protein localizedpredominantly to the nucleus (Fig. 6E). In contrast, in cells trans-fected with 3�FLAG-CMV-Wwtr1 only, Wwtr1 localized pre-dominantly to the cytoplasm (58%) or was equally distributedbetween the nucleus and cytoplasm (30%) and localized predom-inantly to the nucleus in only 12% of the cells. Coexpression ofboth EGFP-Glis3 and FLAG-Wwtr1 had little impact on thedistribution of Glis3. However, the percentage of cells in whichWwtr1 predominantly localized to the nucleus increased dramat-ically (from 12 to 61%), while a concomitant decline was observedin the percentage of cells in which Wwtr1 predominantly localizedto the cytoplasm (Fig. 6F). In contrast to what occurred with

EGFP-Glis3, coexpression of EGFP-Glis3M4 and Wwtr1 did notchange the subcellular distribution of Wwtr1. These observationsare consistent with the concept that Glis3 and Wwtr1 interact witheach other in a P/LPXY-dependent manner and that this inter-action promotes localization or retention of Wwtr1 to the nucleus(Fig. 6E and F). Glis3�ZF5, which is predominantly localized tothe nucleus, was also able to enhance the nuclear localization ofWwtr1, supporting our conclusion that it is able to interact withWwtr1 (Fig. 6E and F).

Wwtr1 functions as a coactivator of Glis3. Previous studies(3, 28) demonstrated that Glis3 can function as an activator oftranscription by binding a specific DNA sequence (Glis-BS) inthe regulatory regions of target genes. To determine whetherWwtr1 is able to modulate Glis3-regulated gene transcription,we analyzed the effect of Wwtr1 on (Glis-BS)6-dependent tran-scriptional activation of the Luc reporter by Glis3. Figure 7A

FIG. 7. Wwtr1 enhances the transcription activity of Glis3. (A) The M4 mutation abolishes the transcription activity of Glis3. The location ofthe activation domain and the PPHY motif are indicated in the schematic view of Glis3 as in Fig. 6A. Cells were cotransfected with (GlisBS)6-Luc,pCMV�, and increasing amounts of p3�FLAG-CMV plasmid expressing Glis3 or Glis3M4, as indicated. After 30 h, the relative Luc activity wasdetermined. Lower panel: the amount of Glis3 protein was examined by Western blot analysis using anti-FLAG M2 antibody. (B) COS-1 cells weretransfected with (GlisBS)6-Luc, pCMV�, p3�FLAG-CMV expressing Glis1, Glis2, or Glis3, and increasing amounts of pCMV-Myc-Wwtr1 asindicated. Cells were collected 24 h later and assayed for reporter activity. The relative Luc activity (normalized to �-galactosidase) was plotted.



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shows that increased Wwtr1 expression significantly enhancedthe induction of Luc reporter activity by Glis3, indicating thatWwtr1 functions as a coactivator of Glis3. The closely relatedYes kinase-associated protein was also able to enhance Glis3activity (data not shown).

We previously showed that the transactivation domain ofGlis3 is located within the 230 amino acids at its C terminus, aregion that encompasses the C-terminal P/LPXY motif (Fig.6A) (3, 28). We therefore examined the effect of the P841A,Y843A double point mutation on the transcriptional activity ofGlis3. As shown in Fig. 7A, these point mutations totally abol-ished the transcriptional activity of Glis3, indicating that thismotif is critical for the transcriptional activity of Glis3 and partof its transactivation domain.

Wwtr1 has little effect on Glis1 and Glis2. To determinewhether Wwtr1 was able to interact with other members of theGlis subfamily, we analyzed the sequence of Glis1 and Glis2for the presence of P/LPXY motifs. This analysis identified twoputative PPXY motifs in Glis1, while no such consensus motifwas present in Glis2. First, we compared the effects of Wwtr1on the transcriptional activities of Glis1-3. Figure 7B showsthat Wwtr1 had only a minor effect on Glis1 transcriptionalactivity, whereas it had no apparent effect on the transcrip-tional activity of Glis2. These observations suggest that Wwtr1functions only as an effective coactivator of Glis3 and does notsignificantly affect the transcriptional activity of Glis1 or Glis2.Next, we examined the interaction between Wwtr1 and Glis1-3by coimmunoprecipitation analysis. These data showed thatWwtr1 formed a complex with Glis3 but not with Glis1 or Glis2(see Fig. S7 in the supplemental material).

DISCUSSION

Recently, mutations in human GLIS3 have been linked toNDH (45, 47). In addition to neonatal diabetes and congenitalhypothyroidism, NDH is associated with facial anomalies,PKD, congenital glaucoma, and liver fibrosis. To further studythe role of Glis3 in this disease, we generated for the first timemutant mice that are impaired in Glis3 function. Glis3 mutantmice develop polycystic kidneys and neonatal diabetes andexhibit a very short life span. The development of hyperglyce-mia and hypoinsulinemia, caused by an insufficiency of pan-creatic � cells (Kang et al., submitted), rather than the devel-opment of polycystic kidneys, which at PND3 is rathermoderate in severity, may be the major cause of the shortenedlife span in Glis3zf/zf mice. Whether these mice develop hypo-thyroidism, glaucoma, and liver fibrosis has yet to be deter-mined. Development of polycystic kidneys was reported onlyfor NDH patients with the most severe abnormalities (NDH1patients) (45). As with Glis3zf/zf mice, NDH1 patients have avery short life span (10 days to 16 months) and neonatal dia-betes. NDH1 patients have a frameshift mutation that resultsin a loss of the C-terminal activation domain of GLIS3 (3, 45).Thus, the phenotype of Glis3zf/zf mutant mice appears to be themost similar to the abnormalities observed in NDH1 patients.This analogy suggests that Glis3zf/zf mutant mice will providean excellent model to study this syndrome at a molecular andmechanistic level.

Development of renal cysts in Glis3zf/zf mice was observed asearly as E15.5 and increased with age. Cyst formation origi-

nated from glomeruli, tubules, and collecting ducts, corre-sponding to the observed expression of Glis3 in kidney inparietal cells and epithelial cells lining the tubules and collect-ing ducts. Thus, Glis3 expression overlaps that of many othergenes implicated in PKD. Study of cystic renal diseases inhumans and in transgenic mice revealed that many proteinsimplicated in these diseases localize to the primary cilium andare either a structural component of the primary cilium orfunction as part of a signal transduction pathway associatedwith the primary cilium (36, 49, 53, 54). These observations ledto the hypothesis that ciliary dysfunction plays a key role in theetiology of cystic renal disease. This link raised the possibilitythat the development of renal cysts in Glis3zf/zf mice may alsohave a connection to the primary cilium. This hypothesis wassupported by our data showing that Glis3 is associated with theprimary cilium in renal tubules and in confluent TKPTS renaltubule epithelial cells expressing EGFP-Glis3. Interestingly,the family member Glis2, which has been implicated innephronophthisis, an autosomal recessive cystic kidney disease(2, 7, 26), was also found to be associated with the primarycilium. Moreover, members of the closely related Gli familyhave been reported to localize to the primary cilium as well(14, 40, 44). The primary cilium plays a critical role in theactivation of the sonic hedgehog (Shh)/Gli signal transductionpathway. In the absence of Shh, its receptor Patched-1 (Ptch1)prevents the accumulation of smoothened (Smo) in the pri-mary cilium. Binding of Shh to Ptch1 inactivates the receptorand results in the activation of Smo, which then accumulateswithin the cilium and activates Gli. Subsequently, Gli translo-cates to the nucleus, where it regulates the transcription oftarget genes. In addition to Shh/Gli, the primary cilium plays acritical role in several other signal transduction pathways (10,12, 54). Its association with the primary cilium suggests thatGlis3 may be part of a cilium-mediated signal transductionpathway and requires activation before it is translocated to thecytoplasm and nucleus (Fig. 8). Because Shh does not affectGlis3-mediated transactivation (our unpublished observa-tions), regulation of Glis3 activity likely involves anothersignal.

Although many proteins associated with the primary ciliumhave been implicated in the development of cystic renal dis-ease, the precise molecular mechanisms underlying these dis-eases have not yet been fully established. Planar cell polarityand oriented cell division have been reported to play an im-portant role in the postnatal development of nephrons, whilemisoriented cell division appears to be part of the mechanismleading to cystogenesis (6, 11, 41, 43, 46). It has been proposedthat the primary cilium may guide oriented cell division (11, 41,43). Our observations indicate that Glis3 dysfunction or Glis3knockdown does not prevent the formation of the primarycilium, suggesting that Glis3 does not regulate the expressionof an essential structural component of the cilium. However,dilated and cystic tubules in Glis3zf/zf mice contain relativelyfewer cells with a primary cilium. Because cell proliferationnegatively affects the formation of primary cilia, this decreasemight involve an indirect mechanism and be due to increasedproliferation, as shown by the increase in BrdU-positive cellsand the reduced level of cdkn1a observed in renal cysts ofGlis3zf/zf mice. In addition to increased proliferation, renaltubule epithelial cells in Glis3zf/zf mice exhibit changes in cell-



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cell and cell-matrix interactions, as indicated by alterations inthe distribution of �-catenin, ZO-1, and Na�-K� ATPase �.Changes in the distribution patterns of EGFR, ZO-1, andE-cadherin were also observed in mouse proximal tubuleTKPTS cells in which Glis3 was downregulated. Interestingly,the effects of Glis3 on junctional proteins resemble those ofPC-1. Expression of PC-1 (Pkd1) in MDCK cells induces cellmigration and reabsorption of ZO-1, E-cadherin, and �-cate-nin at the leading edge of migrating cells, while Pkd1/

mouse embryo fibroblasts exhibit a reduced migratory capabil-ity (8). It was concluded that PC-1 might be a regulator ofepithelial plasticity by controlling cell polarity, cell migration,and cell-cell and cell-matrix interactions, functions importantin the formation, elongation, and maintenance of renal tu-bules. Such a mechanism may also play a role in the control ofrenal functions by Glis3. Interestingly, the observation thatDctn5 and Pkd1 expression were decreased in the kidneys ofGlis3zf/zf mice and in Glis3 downregulated TKPTS cells wouldbe consistent with this hypothesis (Fig. 5). Also notable wasthat in Glis3zf/zf mice, pancreatic tubules were also found to bedilated, suggesting a common mechanism for renal and pan-creatic cyst formation (Kang et al., submitted). Whether such

a common mechanism is responsible for the insufficiency inpancreatic � cells observed in Glis3zf/zf mice needs furtherstudy.

Because of the significant similarities between different cys-tic renal phenotypes and the connection between ciliary pro-teins and cystic renal diseases (49, 53), it is not surprising thatfunctional links are being found between some of the proteinsimplicated in these diseases. For example, PKHD1 is a targetgene of hepatocyte nuclear factor HNF-1�, and mutations inboth genes have been implicated in cystic renal disease (5, 13,16, 36, 49). In this study, we identify a novel link between Glis3and the transcriptional modulator Wwtr1. In both Glis3mutant mice and Wwtr1 null mice, the development of glo-merulocysts are prominent (20, 34, 48) and both Glis3 andWwtr1 promote osteogenesis and repress adipogenesis (4, 19).Wwtr1 can function as a corepressor as well as a coactivator(19). It represses peroxisome proliferator-activated receptor �(PPAR�)-mediated transcription while it enhances transcrip-tional activation by Cbfa1/Runx2, T-box transcription factor 5(TBX5), paired box homeotic gene 3 (Pax3), and thyroid tran-scription factor 1 (TTF1) (9, 18, 38, 42). Wwtr1 interactsthrough its WW domain directly with these transcription fac-tors by recognizing a P/LPXY motif. Glis3, which functions asa positive regulator of Glis-BS-dependent transcription (3, 4,28), contains four putative P/LPXY motifs (Fig. 6A). Theseobservations raised the possibility that Wwtr1 and Glis3 mightinteract with each other and that Wwtr1 might function as acoactivator of Glis3-mediated transcription. Coimmunopre-cipitation and mammalian two-hybrid analyses demonstratedthat Wwtr1 and Glis3 are part of the same protein complex,while in vitro pull-down analysis indicated that Glis3 interactswith Wwtr1 directly. Deletion of its N terminus affected theinteraction of Glis3 with Wwtr1 to some extent, suggesting thatthe N terminus may contribute to the interaction eitherthrough direct binding or through the mediation of other pro-teins within the Glis3-Wwtr1 complex. Deletion of the C ter-minus, containing the fourth P/LPXY motif, or mutation of theC-terminal motif (Glis3M4) abolished the interaction of Glis3with Wwtr1 almost completely. These data indicate that theP/LPXY motif is a major requirement for the interaction andare consistent with the conclusion that Wwtr1 recognizes Glis3directly via this motif. We further showed that increased ex-pression of Wwtr1 significantly enhanced Glis3-mediated tran-scriptional activation, consistent with the concept that it acts asa coactivator of Glis3. Although the family member Glis1 con-tains two putative P/LPXY motifs, Wwtr1 did not interact withGlis1 and did not significantly enhance Glis1 transcriptionalactivity. Glis2, which does not contain any P/LPXY motif, alsodid not interact with Wwtr1. These observations indicate thatthe interaction of Glis3 with Wwtr1 is specific and that Wwtr1functions as an effective coactivator of Glis3 but not of Glis1 orGlis2. We previously showed that the transcriptional activationdomain is located in the C-terminal region of Glis3, a regionthat includes the fourth P/LPXY motif (3, 28). This, togetherwith the observation that this motif is required for the inter-action with the coactivator Wwtr1, suggested that this motifmay be part of the transactivation function of Glis3. This con-clusion was supported by data showing that mutations in thismotif (PPHY to PAHA) greatly diminished the transcriptionalactivity of Glis3 (Fig. 7A). This is consistent with the finding

FIG. 8. Model illustrating the links between Glis3, the primarycilium, Wwtr1, and cystic renal disease. We demonstrate that Glis3 isassociated with the primary cilium, where it may be activated by ayet-unidentified signal (e.g., flow, G-protein-linked receptor). This ac-tivation may involve phosphorylation of Glis3, changes in protein-protein interaction, and/or proteolytic processing of Glis3. ActivatedGlis3 (Glis3*) is subsequently transported by the intraflagellar trans-port into the cytoplasm and into the nucleus, where it can interact withvarious coactivators (Co-act.), including Wwtr1. Wwtr1 can interactwith other transcription factors (TFs). Thus, Glis3 and Wwtr1 are partof overlapping transcription regulatory networks. Glis3-Wwtr1 andother Glis3-coactivator complexes regulate the expression of Glis3target genes/proteins necessary for the maintenance of normal renalarchitecture and functions. Dysfunction of the Glis3 or Wwtr1 signal-ing pathways results in aberrant gene regulation and the developmentof cystic renal disease.



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that a base insertion in the GLIS3 gene of NDH1 patientsresults in a frameshift and that deletion of the C terminus,including the fourth P/LPXY motif, causes loss of Glis3 tran-scriptional activity (3, 45). Because Glis3�ZF5, which matchesthe mutation in Glis3zf/zf mice, can interact with Wwtr1 but isunable to bind Glis-BS, the development of the renal andpancreatic phenotype in Glis3zf/zf mice is related to its inabilityto induce GLIS-BS-dependent transactivation and not due tolack of Wwtr1 interaction.

The interaction between Wwtr1 and Glis3 was further sup-ported by observations showing that coexpression of Glis3 withWwtr1 promoted the nuclear localization of Wwtr1. Previousstudies reported that Wwtr1 shuttles in and out of the nucleus(22, 31, 51, 55). This shuttling seems to be controlled at severallevels. In the cytoplasm, phosphorylation of Wwtr1 by theHippo kinase signaling cascade promotes interaction with 14-3-3 proteins and retention in the cytoplasm. In the nucleus,Wwtr1 can interact with several transcription factors (9, 18, 38,42, 51). It has been suggested that competition may exist be-tween the transcriptional machinery and 14-3-3 proteins forWwtr1 binding. Interaction of Glis3 with Wwtr1 may result innuclear retention of Wwtr1 (Fig. 6E), as has been proposed forthe interaction between Wwtr1 and the transcription cofactorARC105 (51). However, we cannot rule out that Glis3 forms acomplex with Wwtr1 in the cytoplasm and then translocates tothe nucleus.

In summary, this study demonstrates that Glis3 mutant micehave a very short life span and develop polycystic kidneys andneonatal diabetes, abnormalities that are very similar to thoseobserved in patients with NDH1. Therefore, these mice pro-vide an excellent model to study the molecular mechanismsunderlying this syndrome. We further identify two importantelements of the Glis3 signal transduction pathway. We showthat Glis3 localizes to the primary cilium and propose thatupon activation of Glis3 by a primary cilium-associated signal-ing pathway, Glis3 is transported by intraflagellar transportinto the cytoplasm and subsequently into the nucleus (Fig. 8).In the nucleus, Glis3 interacts with several coactivators, includ-ing Wwtr1, that mediate the transcriptional activation of Glis3target genes. Thus, Glis3 and Wwtr1 are part of overlappingtranscription regulatory networks that are critical in maintain-ing normal renal structure and homeostasis.

ACKNOWLEDGMENTS

We thank Gregory Germino, Johns Hopkins University, for hisadvice on the polycystic kidney phenotype; Darlene Dixon and TinaTeng, NIEHS, for their comments on the manuscript; and RandyThresher, UNC Transgenic Facility, and Laura Miller, NIEHS, fortheir support and advice with generating the mutant mice.

This research was supported by the Intramural Research Program ofthe NIEHS (Z01-ES-101485).

We declare no conflicts of interest.

REFERENCES

1. Aruga, J. 2004. The role of Zic genes in neural development. Mol. Cell.Neurosci. 26:205–221.

2. Attanasio, M., N. H. Uhlenhaut, V. H. Sousa, J. F. O’Toole, E. Otto, K.Anlag, C. Klugmann, A. C. Treier, J. Helou, J. A. Sayer, D. Seelow, G.Nurnberg, C. Becker, A. E. Chudley, P. Nurnberg, F. Hildebrandt, and M.Treier. 2007. Loss of GLIS2 causes nephronophthisis in humans and mice byincreased apoptosis and fibrosis. Nat. Genet. 39:1018–1024.

3. Beak, J. Y., H. S. Kang, Y. S. Kim, and A. M. Jetten. 2008. Functionalanalysis of the zinc finger and activation domains of Glis3 and mutantGlis3(NDH1). Nucleic Acids Res. 36:1690–1702.

4. Beak, J. Y., H. S. Kang, Y. S. Kim, and A. M. Jetten. 2007. Kruppel-like zincfinger protein Glis3 promotes osteoblast differentiation by regulating FGF18expression. J. Bone Miner. Res. 22:1234–1244.

5. Bergmann, C., J. Senderek, F. Kupper, F. Schneider, C. Dornia, E. Wind-elen, T. Eggermann, S. Rudnik-Schoneborn, J. Kirfel, L. Furu, L. F.Onuchic, S. Rossetti, P. C. Harris, S. Somlo, L. Guay-Woodford, G. G.Germino, M. Moser, R. Buttner, and K. Zerres. 2004. PKHD1 mutations inautosomal recessive polycystic kidney disease (ARPKD). Hum. Mutat. 23:453–463.

6. Bhunia, A. K., K. Piontek, A. Boletta, L. Liu, F. Qian, P. N. Xu, F. J.Germino, and G. G. Germino. 2002. PKD1 induces p21(waf1) and regulationof the cell cycle via direct activation of the JAK-STAT signaling pathway ina process requiring PKD2. Cell 109:157–168.

7. Bisgrove, B. W., and H. J. Yost. 2006. The roles of cilia in developmentaldisorders and disease. Development 133:4131–4143.

8. Boca, M., L. D’Amato, G. Distefano, R. S. Polishchuk, G. G. Germino, and A.Boletta. 2007. Polycystin-1 induces cell migration by regulating phosphatidylino-sitol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependentcell cell mechanical adhesion. Mol. Biol. Cell 18:4050–4061.

9. Cui, C. B., L. F. Cooper, X. Yang, G. Karsenty, and I. Aukhil. 2003. Tran-scriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ.Mol. Cell. Biol. 23:1004–1013.

10. Deane, J. A., and S. D. Ricardo. 2007. Polycystic kidney disease and the renalcilium. Nephrology 12:559–564.

11. Fischer, E., E. Legue, A. Doyen, F. Nato, J. F. Nicolas, V. Torres, M. Yaniv,and M. Pontoglio. 2006. Defective planar cell polarity in polycystic kidneydisease. Nat. Genet. 38:21–23.

12. Fliegauf, M., T. Benzing, and H. Omran. 2007. When cilia go bad: ciliadefects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8:880–893.

13. Gresh, L., E. Fischer, A. Reimann, M. Tanguy, S. Garbay, X. Shao, T.Hiesberger, L. Fiette, P. Igarashi, M. Yaniv, and M. Pontoglio. 2004. Atranscriptional network in polycystic kidney disease. EMBO J. 23:1657–1668.

14. Haycraft, C. J., B. Banizs, Y. Aydin-Son, Q. Zhang, E. J. Michaud, and B. K.Yoder. 2005. Gli2 and Gli3 localize to cilia and require the intraflagellartransport protein polaris for processing and function. PLoS Genet. 1:e53.

15. Hiesberger, T., Y. Bai, X. Shao, B. T. McNally, A. M. Sinclair, X. Tian, S.Somlo, and P. Igarashi. 2004. Mutation of hepatocyte nuclear factor-1betainhibits Pkhd1 gene expression and produces renal cysts in mice. J. Clin.Investig. 113:814–825.

16. Hiesberger, T., X. Shao, E. Gourley, A. Reimann, M. Pontoglio, and P.Igarashi. 2005. Role of the hepatocyte nuclear factor-1beta (HNF-1beta)C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cysto-genesis. J. Biol. Chem. 280:10578–10586.

17. Hildebrandt, F., and W. Zhou. 2007. Nephronophthisis-associated ciliopa-thies. J. Am. Soc. Nephrol. 18:1855–1871.

18. Hong, J. H., E. S. Hwang, M. T. McManus, A. Amsterdam, Y. Tian, R.Kalmukova, E. Mueller, T. Benjamin, B. M. Spiegelman, P. A. Sharp, N.Hopkins, and M. B. Yaffe. 2005. TAZ, a transcriptional modulator ofmesenchymal stem cell differentiation. Science 309:1074–1078.

19. Hong, J. H., and M. B. Yaffe. 2006. TAZ: a beta-catenin-like molecule thatregulates mesenchymal stem cell differentiation. Cell Cycle 5:176–179.

20. Hossain, Z., S. M. Ali, H. L. Ko, J. Xu, C. P. Ng, K. Guo, Z. Qi, S. Ponniah,W. Hong, and W. Hunziker. 2007. Glomerulocystic kidney disease in micewith a targeted inactivation of Wwtr1. Proc. Natl. Acad. Sci. USA 104:1631–1636.

21. Igarashi, P., X. Shao, B. T. McNally, and T. Hiesberger. 2005. Roles ofHNF-1beta in kidney development and congenital cystic diseases. KidneyInt. 68:1944–1947.

22. Kanai, F., P. A. Marignani, D. Sarbassova, R. Yagi, R. A. Hall, M. Donowitz,A. Hisaminato, T. Fujiwara, Y. Ito, L. C. Cantley, and M. B. Yaffe. 2000.TAZ: a novel transcriptional co-activator regulated by interactions with14-3-3 and PDZ domain proteins. EMBO J. 19:6778–6791.

23. Kang, H. S., M. Angers, J. Y. Beak, X. Wu, J. M. Gimble, T. Wada, W. Xie,J. B. Collins, S. F. Grissom, and A. M. Jetten. 2007. Gene expressionprofiling reveals a regulatory role for ROR{alpha} and ROR{gamma} inphase I and phase II metabolism. Physiol. Genomics 31:281–294.

24. Kasper, M., G. Regl, A. M. Frischauf, and F. Aberger. 2006. GLI transcrip-tion factors: mediators of oncogenic Hedgehog signalling. Eur. J. Cancer42:437–445.

25. Kim, S. C., Y. S. Kim, and A. M. Jetten. 2005. Kruppel-like zinc fingerprotein Gli-similar 2 (Glis2) represses transcription through interaction withC-terminal binding protein 1 (CtBP1). Nucleic Acids Res. 33:6805–6815.

26. Kim, Y. S., H. S. Kang, R. Herbert, J. Y. Beak, J. B. Collins, S. F. Grissom,and A. M. Jetten. 2008. Kruppel-like zinc finger protein Glis2 is essential forthe maintenance of normal renal functions. Mol. Cell. Biol. 28:2358–2367.

27. Kim, Y. S., M. Lewandoski, A. O. Perantoni, S. Kurebayashi, G. Nakanishi,and A. M. Jetten. 2002. Identification of Glis1, a novel Gli-related, Kruppel-like zinc finger protein containing transactivation and repressor functions.J. Biol. Chem. 277:30901–30913.

28. Kim, Y. S., G. Nakanishi, M. Lewandoski, and A. M. Jetten. 2003. GLIS3, anovel member of the GLIS subfamily of Kruppel-like zinc finger proteinswith repressor and activation functions. Nucleic Acids Res. 31:5513–5525.



cb.asm.org/

Dow

nloaded from

http://mcb.asm.org/

29. Kolb, R. J., and S. M. Nauli. 2008. Ciliary dysfunction in polycystic kidneydisease: an emerging model with polarizing potential. Front. Biosci. 13:4451–4466.

30. Lamar, E., C. Kintner, and M. Goulding. 2001. Identification of NKL, anovel Gli-Kruppel zinc-finger protein that promotes neuronal differentia-tion. Development 128:1335–1346.

31. Lei, Q. Y., H. Zhang, B. Zhao, Z. Y. Zha, F. Bai, X. H. Pei, S. Zhao, Y. Xiong,and K. L. Guan. 2008. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell.Biol. 28:2426–2436.

32. Lin, F., T. Hiesberger, K. Cordes, A. M. Sinclair, L. S. Goldstein, S. Somlo,and P. Igarashi. 2003. Kidney-specific inactivation of the KIF3A subunit ofkinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease.Proc. Natl. Acad. Sci. USA 100:5286–5291.

33. Liu, A., B. Wang, and L. A. Niswander. 2005. Mouse intraflagellar transportproteins regulate both the activator and repressor functions of Gli transcrip-tion factors. Development 132:3103–3111.

34. Makita, R., Y. Uchijima, K. Nishiyama, T. Amano, Q. Chen, T. Takeuchi, A.Mitani, T. Nagase, Y. Yatomi, H. Aburatani, O. Nakagawa, E. V. Small, P.Cobo-Stark, P. Igarashi, M. Murakami, J. Tominaga, T. Sato, T. Asano, Y.Kurihara, and H. Kurihara. 2008. Multiple renal cysts, urinary concentra-tion defects, and pulmonary emphysematous changes in mice lacking TAZ.Am. J. Physiol. Renal Physiol. 294:F542–F553.

35. Menezes, L. F., Y. Cai, Y. Nagasawa, A. M. Silva, M. L. Watkins, A. M. DaSilva, S. Somlo, L. M. Guay-Woodford, G. G. Germino, and L. F. Onuchic.2004. Polyductin, the PKHD1 gene product, comprises isoforms expressed inplasma membrane, primary cilium, and cytoplasm. Kidney Int. 66:1345–1355.

36. Menezes, L. F., and L. F. Onuchic. 2006. Molecular and cellular pathogen-esis of autosomal recessive polycystic kidney disease. Braz. J. Med. Biol. Res.39:1537–1548.

37. Murakami, M., M. Nakagawa, E. N. Olson, and O. Nakagawa. 2005. A WWdomain protein TAZ is a critical coactivator for TBX5, a transcription factorimplicated in Holt-Oram syndrome. Proc. Natl. Acad. Sci. USA 102:18034–18039.

38. Murakami, M., J. Tominaga, R. Makita, Y. Uchijima, Y. Kurihara, O.Nakagawa, T. Asano, and H. Kurihara. 2006. Transcriptional activity of Pax3is co-activated by TAZ. Biochem. Biophys. Res. Commun. 339:533–539.

39. Nakashima, M., N. Tanese, M. Ito, W. Auerbach, C. Bai, T. Furukawa, T.Toyono, A. Akamine, and A. L. Joyner. 2002. A novel gene, GliH1, withhomology to the Gli zinc finger domain not required for mouse development.Mech. Dev. 119:21–34.

40. Oro, A. E. 2007. The primary cilia, a ‘Rab-id’ transit system for hedgehogsignaling. Curr. Opin. Cell Biol. 19:691–696.

41. Pan, J., and W. Snell. 2007. The primary cilium: keeper of the key to celldivision. Cell 129:1255–1257.

42. Park, K. S., J. A. Whitsett, T. Di Palma, J. H. Hong, M. B. Yaffe, and M.Zannini. 2004. TAZ interacts with TTF-1 and regulates expression of sur-factant protein-C. J. Biol. Chem. 279:17384–17390.

43. Quarmby, L. M., and J. D. Parker. 2005. Cilia and the cell cycle? J. Cell Biol.169:707–710.

44. Rohatgi, R., and M. P. Scott. 2007. Patching the gaps in Hedgehog signalling.Nat. Cell Biol. 9:1005–1009.

45. Senee, V., C. Chelala, S. Duchatelet, D. Feng, H. Blanc, J. C. Cossec, C.Charon, M. Nicolino, P. Boileau, D. R. Cavener, P. Bougneres, D. Taha, andC. Julier. 2006. Mutations in GLIS3 are responsible for a rare syndrome withneonatal diabetes mellitus and congenital hypothyroidism. Nat. Genet. 38:682–687.

46. Simons, M., and G. Walz. 2006. Polycystic kidney disease: cell divisionwithout a c(l)ue? Kidney Int. 70:854–864.

47. Taha, D., M. Barbar, H. Kanaan, and J. Williamson Balfe. 2003. Neonataldiabetes mellitus, congenital hypothyroidism, hepatic fibrosis, polycystic kid-neys, and congenital glaucoma: a new autosomal recessive syndrome? Am. J.Med. Genet. 122A:269–A273.

48. Tian, Y., R. Kolb, J.-H. Hong, J. Carroll, D. Li, J. You, R. Bronson, M. B.Yaffe, J. Zhou, and T. Benjamin. 2007. TAZ promotes PC2 degradationthrough a SCF�-Trcp E3 ligase complex. Mol. Cell. Biol. 27:6383–6395.

49. Torres, V. E., and P. C. Harris. 2006. Mechanisms of disease: autosomaldominant and recessive polycystic kidney diseases. Nat. Clin. Pract. Nephrol.2:40–55.

50. Uhlenhaut, N. H., and M. Treier. 2008. Transcriptional regulators in kidneydisease: gatekeepers of renal homeostasis. Trends Genet. 24:361–371.

51. Varelas, X., R. Sakuma, P. Samavarchi-Tehrani, R. Peerani, B. M. Rao, J.Dembowy, M. B. Yaffe, P. W. Zandstra, and J. L. Wrana. 2008. TAZ controlsSmad nucleocytoplasmic shuttling and regulates human embryonic stem-cellself-renewal. Nat. Cell Biol. 10:837–848.

52. Ward, C. J., D. Yuan, T. V. Masyuk, X. Wang, R. Punyashthiti, S. Whelan,R. Bacallao, R. Torra, N. F. LaRusso, V. E. Torres, and P. C. Harris. 2003.Cellular and subcellular localization of the ARPKD protein; fibrocystin isexpressed on primary cilia. Hum. Mol. Genet. 12:2703–2710.

53. Wilson, P. D. 2004. Polycystic kidney disease: new understanding in thepathogenesis. Int. J. Biochem. Cell Biol. 36:1868–1873.

54. Yoder, B. K. 2007. Role of primary cilia in the pathogenesis of polycystickidney disease. J. Am. Soc. Nephrol. 18:1381–1388.

55. Zeng, Q., and W. Hong. 2008. The emerging role of the hippo pathway in cellcontact inhibition, organ size control, and cancer development in mammals.Cancer Cell 13:188–192.

56. Zhang, F., and A. M. Jetten. 2001. Genomic structure of the gene encodingthe human GLI-related, Kruppel-like zinc finger protein GLIS2. Gene 280:49–57.

57. Zhang, F., G. Nakanishi, S. Kurebayashi, K. Yoshino, A. Perantoni, Y. S.Kim, and A. M. Jetten. 2002. Characterization of Glis2, a novel gene encod-ing a Gli-related, Kruppel-like transcription factor with transactivation andrepressor functions. Roles in kidney development and neurogenesis. J. Biol.Chem. 277:10139–10149.



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Glis3 is associated with primary cilia and Wwtr1/TAZ and implicated in polycystic kidney disease