Characterization of the human Suppressor of fused, a ... · factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well

INTRODUCTION

The Hedgehogs (HH) constitute a family of secreted proteinswhich play important roles in tissue patterning duringearly embryogenesis in vertebrates and invertebrates(Hammerschmidt et al., 1997; Ingham, 1995). DrosophilaHedgehog (Hh) is required for proper segmentation of thelarvae, and for growth and organization of the wing and otherappendages in the adult fly. The mammalian HH proteins,which include Sonic hedgehog (Shh), Indian hedgehog (Ihh)and Desert hedgehog (Dhh), are expressed in a tissue-specificmanner. They control the specification of ventral cell types inthe central nervous system, left-right asymmetry, growth andpatterning of the somites and limbs, cartilage differentiation,organogenesis and spermatogenesis. Mutations in genes thatmediate the HH signal have been linked to human cancer anddevelopmental disorders (reviewed in Johnson and Scott,1998), thus establishing an important role for this pathway innormal cell growth control.

The mechanism of Hh signal transduction is not fullyunderstood. However, genetic studies in Drosophila haveidentified a diverse array of transmembrane and intracellularproteins which serve as specific components in the Hhsignaling pathway (reviewed in Ingham, 1998; Johnson andScott, 1998; Tabin and McMahon, 1997). The pathwayculminates in the activation of Cubitus interruptus (Ci)(Alexandre et al., 1996; Dominguez et al., 1996), a zinc fingertranscription factor homologous to vertebrate Gli proteins(Orenic et al., 1990). Conservation of Hh signal transductionmechanisms is suggested by the ability of ectopicallyexpressed Xenopus or human Gli (hGli) to mimic Shh in theinduction of floor-plate specific markers and ventral neuronalcell types, both in frog (Lee et al., 1997) and mouse (Hynes etal., 1997). Likewise, the cyclic AMP-dependent protein kinase(PKA) exerts a common negative regulatory effect on HHsignaling in both flies (Jiang and Struhl, 1995; Li et al., 1995)and rodents (Epstein et al., 1996; Fan et al., 1995;Hammerschmidt et al., 1996; Hynes et al., 1995). Moreover,

4437Journal of Cell Science 112, 4437-4448 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0508

Drosophila Suppressor of fused (Su(fu)) encodes a novel468-amino-acid cytoplasmic protein which, by geneticanalysis, functions as a negative regulator of the Hedgehogsegment polarity pathway. Here we describe the primarystructure, tissue distribution, biochemical and functionalanalyses of a human Su(fu) (hSu(fu)). Two alternativelyspliced isoforms of hSu(fu) were identified, predictingproteins of 433 and 484 amino acids, with a calculatedmolecular mass of 48 and 54 kDa, respectively. The twoproteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Bothisoforms were expressed in multiple embryonic and adulttissues, and exhibited a developmental profile consistentwith a role in Hedgehog signaling. The hSu(fu) contains ahigh-scoring PEST-domain, and exhibits an overall 37%sequence identity (63% similarity) with the Drosophilaprotein and 97% sequence identity with the mouse Su(fu).The hSu(fu) locus mapped to chromosome 10q24-q25, aregion which is deleted in glioblastomas, prostate cancer,

malignant melanoma and endometrial cancer. HSu(fu) wasfound to repress activity of the zinc-finger transcriptionfactor Gli, which mediates Hedgehog signaling invertebrates, and to physically interact with Gli, Gli2 andGli3 as well as with Slimb, an F-box containing proteinwhich, in the fly, suppresses the Hedgehog response, in partby stimulating the degradation of the fly Gli homologue.Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our dataprovide biochemical and functional evidence for thehypothesis that Su(fu) is a key negative regulator in thevertebrate Hedgehog signaling pathway. The data furthersuggest that Su(fu) can act by binding to Gli and inhibitingGli-mediated transactivation as well as by serving as anadaptor protein, which links Gli to the Slimb-dependentproteasomal degradation pathway.

Key words: Hedgehog, In situ hybridization, Chromosomallocalization, Trans-activation

SUMMARY

Characterization of the human Suppressor of fused, a negative regulator of

the zinc-finger transcription factor Gli

Donna M. Stone1, Maximilien Murone2, Shiuh-Ming Luoh2, Weilan Ye1, Mark P. Armanini1, Austin Gurney3,Heidi Phillips1, Jennifer Brush3, Audrey Goddard3, Frederic J. de Sauvage2 and Arnon Rosenthal1,*

Departments of 1Neuroscience, 2Molecular Oncology and 3Molecular Biology, Genentech, Inc. 1 DNA Way, South San Francisco,CA 94080, USA*Author for correspondence (e-mail: [email protected])

Accepted 22 September; published on WWW 17 November 1999

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vertebrate homologues have been identified for DrosophilaPatched (Goodrich et al., 1996), a multi-pass transmembraneprotein which, by genetic analysis, functions downstream ofHh to inhibit signaling (Alcedo et al., 1996; Hooper and Scott,1989), and Drosophila Smoothened (Smo; Stone et al., 1996),a seven-pass transmembrane protein absolutely required fortransduction of the Hh signal (Alcedo et al., 1996; Hooper,1994). A combination of biochemical data obtained invertebrate systems and genetic analyses in Drosophila predictPatched to be the ligand-binding component and Smo thesignaling component, in a multi-subunit receptor complex forHH proteins (Chen and Struhl, 1998; Marigo et al., 1996;Murone et al., 1999; Stone et al., 1996). Taken together, thisevidence for evolutionary conservation suggests that otheridentified members of the Drosophila signaling pathway maylikely have vertebrate counterparts.

Drosophila Suppressor of fused (dSu(fu)) is a novelcytoplasmic PEST-containing protein (Pham et al., 1995)which, when mutated in a wild-type background, confers amild phenotype suggestive of constitutive Hh signaling(Ohlmeyer and Kalderon, 1998). Moreover, the same mutationcan fully suppress both embryonic and adult phenotypesresulting from mutations in Fused (Fu) (Preat, 1992), a serine-threonine kinase required for Hh signaling (Mariol et al.,1987). DSu(fu) interacts physically with Fu and Ci (Monnieret al., 1998), and the latter interaction has been hypothesizedto maintain Ci in an inactive state by sequestering it in thecytoplasm and/or by preventing its processing to an active form(Ohlmeyer and Kalderon, 1998). In the absence of Hhsignaling, full-length Ci is proteolytically cleaved to producean amino-terminal 75-kDa transcriptional repressor form (Aza-Blanc et al., 1997), presumably through targeting of PKA-phosphorylated Ci (Chen et al., 1998) to the ubiquitin-proteasome pathway by the F-box containing protein, Slimb(Jiang and Struhl, 1998). Reception of the Hh signal ispredicted to activate Fu, inactivate dSu(fu) and triggerdownstream events culminating in the conversion of Ci into atransactivator of Hh target genes.

To gain further biochemical and functional insight into therole of Su(fu) in HH signaling, we have cloned two isoformsof a human homologue of this protein and examined theexpression pattern of Su(fu) during development and in theadult. Additionally, we studied physical interactions betweenhSu(fu) and other signaling components in the HH pathway,including members of the vertebrate Gli protein family and avertebrate Slimb homologue, and analyzed the functionalimplications of these interactions.

MATERIALS AND METHODS

Cloning and sequencing of the human Su(fu) cDNAHuman fetal lung and human fetal testis libraries prepared in a CMV-based mammalian expression vector (pRK) were probed withan oligonucleotide derived from a mouse EST (accession no.AA061391; 5′GAGCACTGGCACTACATCAGCTTTGGCCTGAG-TGATCTCT3′). Several positive clones were selected and analyzedby restriction digest; two clones which exhibited different restrictionpatterns were sequenced on both strands by standard protocols.

Subcloning and production of cDNA constructsTwo different hSu(fu) cDNAs were identified, hSu(fu)433 and

hSu(fu)484, which differed in sequence at the 3′ end of the openreading frame (ORF). By PCR, the hSu(fu)433 cDNA was epitope-tagged with the flag peptide at its carboxy terminus, to produce pRK-hSu(fu)433. pRK-hSu(fu)484 was tagged in a similar manner, and the5′ end (to the XhoI site at base 946 of hSu(fu)433) was replaced withthe 5′ end of hSu(fu)433, since the hSu(fu)484 cDNA was missing458-bp of 5′ sequence. The human Gli cDNA (provided by Dr KenKinzler) was cloned into the same expression vector, and a 9E10 c-myc epitope was introduced at the amino terminus (immediately afterthe first ATG), to produce pRK-hGli. Human Gli3 (provided by DrMike Ruppert) was also cloned into pRK, to produce pRK-hGli3. Thecoding region of mouse Gli2 was obtained by PCR with Takara LApolymerase (Takara Shuzo Co., Ltd.) using Marathon Ready mouseE11 cDNA (Clontech) as template, and was cloned into pRK, yieldingpRK-mGli2. A partial mouse Slimb cDNA was obtained fromGenome Systems (IMAGE clone #1068742) and extended by 5′RACE. Several different 5′ RACE products were recovered,suggesting that the gene is subject to alternative splicing at its 5′ end(see also Theodosiou et al., 1998; Margottin et al., 1998). Thesequence most closely matching the amino terminus of human Slimb(Theodosiou et al., 1998) was isolated and an HA tag was introducedat the amino terminus to produce pRK-Slimb. Mouse Slimb differedfrom human Slimb at 9 out of 572 amino acids. pRK-Slimb∆F wasgenerated by deleting amino acids 1-207, which encompassed the F-box motif, and adding an HA tag at the amino terminus. Theglutathione-S-transferase (GST)-hSu(fu) expression construct(pGEX-hSu(fu)) was made by fusing the hSu(fu)433 cDNA in-frameto the carboxy terminus of GST in a pGEX vector (Pharmacia).

Chromosomal localization of the hSu(fu) geneHuman metaphase chromosome spreads from cultured bloodlymphocytes were prepared by standard procedures, and subject tofluorescence in situ hybridization (FISH) as described (Heng et al.,1992; Heng and Tsui, 1993), using the entire hSu(fu)433 cDNA asprobe. FISH signals and DAPI banding patterns of each chromosomalspread were recorded separately, then superimposed to assign thehSu(fu) mapping position.

Northern blot analysis and semi-quantitative PCRHuman multiple tissue northern blots (Clontech) were hybridized witha 32P-labelled SmaI-XhoI 853-bp fragment of hSu(fu)433 cDNAaccording to the manufacturer’s protocol, and washed to a stringencyof 0.2×SSC, 0.1% SDS at 65°C, prior to exposure to X-ray film. Semi-quantitative PCR was performed using 10 pmol each of primers: P1,5′-CCAATCAACCCTCAGCGGCAGAATG-3′; P2, 5′-CGAGGC-CAGCAGCTCGTTC-3′; and P3, 5′-GTAGGTGAGAAAGAG-GGCTGTC-3′, in a standard 25 µl PCR reaction containing 100 µMdNTPs, 0.25 µl [33P]dATP (10 mCi/ml), and 50 ng plasmid DNA fromhuman tissue cDNA expression libraries as template. DNA wasamplified by Taq DNA Polymerase for 30 cycles, then 10 µl was runon a 4% TBE acrylamide gel, which was dried and exposed to X-rayfilm.

In situ hybridization to Su(fu) mRNAWhole-mount in situ hybridization to embryonic day 8.5 (E8.5) mouseembryos was performed as described (Shimamura and Rubenstein,1997). The probe was a digoxigenin-labeled RNA, synthesized withT7 RNA polymerase and a mouse Su(fu) cDNA PCR template,corresponding to nucleotides 116-390 (nucleotide 1=A in the initiatorATG) of the human sequence. For in situ hybridization to tissuesections, rat E11.5 and E15.5 whole embryos, and postnatal day 1 (P1)rat brains were immersion-fixed overnight at 4°C in 4%paraformaldehyde, cryoprotected overnight in 15% sucrose,embedded in OTC (VWR Scientific), and frozen on liquid nitrogen.Adult rat brains were fresh frozen with powdered dry ice. Adult ratspinal cord and mouse testis were embedded in OTC and frozen onliquid nitrogen. Sections were cut at 16 µm, and processed for in situ

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4439Human Su(fu) negatively regulates Gli

hybridization as described previously (Phillips et al., 1990).[33P]UTP-labeled RNA probes were generated as described (Meltonet al., 1984). Sense and antisense probes were synthesized with T7RNA polymerase from a hSu(fu)433 cDNA PCR fragmentencompassing nucleotides 97-424 of the human sequence.

ImmunocytochemistryC3H10T1/2 or COS-7 cells were maintained in DMEM with 10%fetal bovine serum. Subconfluent cultures in ProNectin F(Stratagene)-coated glass chamber slides were transientlycotransfected with pRK-hGli, pRK-hSu(fu)484 or pRK-hSu(fu)433using lipofectamine (Gibco BRL). 24 hours later, cells were fixed in4% paraformaldehyde for 10 minutes, with or without prior detergentextraction (0.1% NP-40 in stabilization buffer: 4% PEG 8000, 1 mMEGTA, 100 mM Pipes, pH 6.9) for 10 minutes at 37°C. For tubulinstaining, proteins were cross-linked with 1 mM DSP (Pierce) instabilization buffer for 10 minutes at 37°C prior to fixation. Cells werepermeabilized in 0.1% Triton-X 100 for 5 minutes, blocked in blockbuffer (5% goat serum in PBS) for 30 minutes, and reacted with

primary antibodies consisting of rabbit anti-hSu(fu) (3 µg/ml; seebelow), anti-c-myc monoclonal (Genentech; 3 µg/ml in block bufferfor 1 hour), or monoclonal anti-β-tubulin (Sigma, clone TUB 2.1;1:250 dilution). Cells were washed and double-labeled with cy3-anti-rabbit IgG (1:350) and cy2-anti-mouse IgG (1:100; JacksonImmunoResearch) for 1 hour in block buffer. Slides were washed andcoverslips attached with Fluoromount-G (Southern BiotechnologyAssoc., Inc.). The hSu(fu) polyclonal antibody was produced byimmunization of rabbits with purified GST-hSu(fu) fusion protein.Resultant antibodies were purified by affinity chromatography on aProtein A column.

In vitro coimmunoprecipitation assay293 cells were grown in DMEM containing 10% fetal bovine serum,to 70% confluence in 10-cm tissue culture dishes. Cells weretransfected with lipofectamine according to the manufacturer’sprotocol (Gibco BRL) using a total of 8 µg of DNA/dish; forcotransfections, 4 µg of each were used. 24 hours later, cells werewashed in PBS (4°C) and lysed directly in 1 ml ice-cold lysis buffer

Fig. 1. Alignment of the predicted protein sequences of human (h), mouse (m) and Drosophila (d) Su(fu). Identical residues are boxed, solidgray regions indicate conserved potential protein kinase C phosphorylation sites, asterisks indicate a conserved potential casein kinase IIphosphorylation site, outlined letters indicate candidate PKA phosphorylation sites, black background with white text indicates highest scoringPEST domains in each sequence (score=26.29 and 26.66 for human and mouse, respectively, and 1.48 for Drosophila Su(fu)). Black baroverlies the 52-amino acid extension in hSu(fu)484. Boxed residue (I at position 433) is a leucine in hSu(fu)433, which immediately precedesthe stop codon. Sequences were aligned by the Clustal algorithm. Needlemen-Wench scoring revealed a 37% identity, 63% similarity, betweenhuman and Drosophila proteins, and 97% identity, 99% similarity between the long form of human and mouse proteins.

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(containing 20 mM Hepes, pH 8.0, 150 mM NaCl, 1% NP-40, 5µg/ml each leupeptin and aprotinin, 1 mM PMSF and 250 µMorthovanadate). Lysate was rotated at 4°C for 20 minutes,centrifuged at 14000 rpm for 20 minutes, and the supernatantsubjected to immunoprecipitation with either 2 µl anti-flag M2monoclonal antibody (Kodak IBI) or 2 µl anti-myc monoclonalantibody (9E10; Genentech) overnight (4°C). In some cases,ethidium bromide was added to the lysates prior toimmunoprecipitation to preclude the possibility of DNA-dependent protein associations (Lai et al., 1992). Protein Asepharose (Pharmacia) was added (25 µl) for 1 hour at 4°C, thebeads were washed 3× with lysis buffer and 2× with 1 M NaCl,and samples were heated in SDS-loading buffer to 70°C for 10minutes. Samples were electrophoresed on 4-12% NuPAGEdenaturing SDS gels (Novex), and proteins detected by blottingto nitrocellulose and probing with antibodies to flag or mycepitopes, using the ECL detection system (Amersham).

GST-fusion protein in vitro binding assaypGEX-hSu(fu) was transformed into DH12S bacterial cells(Gibco BRL), and a 500-ml overnight culture was processed forpurification of GST-hSu(fu) fusion protein according to themanufacturer’s protocol (Pharmacia). Fusion protein was eluted

from the beads with excess reduced glutathione, andeluted protein was quantified by OD280 measurementand visualization on denaturing SDS-polyacrylamidegel (data not shown). Glutathione-sepharose beadswere loaded with 4 µg fusion protein or GST (Sigma)for 2 hours at 4°C, then washed 3× with bindingbuffer. Beads (25 µl of a 50:50 slurry) were incubatedwith 2-8 µl of 35S-labeled in vitro-translated hGli,mGli2, hGli3, mSlimb or hSu(fu)433 in 50 µl bindingbuffer for 2 hours at 4°C. The beads were washed 3×with lysis buffer, and processed for SDS-PAGE. Gelswere fixed, amplified in EN3HANCE (Dupont NEN),dried and exposed to Kodak X-AR film. Bindingbuffer was 50 mM Tris-HCl, pH 8.0, 150 mM NaCland protease inhibitors (as above). pRK-hGli, pRK-

D. M. Stone and others

Fig. 2. Chromosomal localization of the human Su(fu) gene.(A) Upper panel shows FISH localization of the biotinylated hSu(fu)probe. Assignment to the long arm of chromosome 10 wasaccomplished by superimposing a DAPI-stained image of the samemitotic figure (lower panel). (B) Diagram of FISH mapping results.Each dot represents double FISH signals on a single chromosomespread. Of a total of 100 cells analyzed, 72 were specifically labeled.

Fig. 3. Northern blot and PCR analysis of hSu(fu)mRNA expression in fetal and adult human tissues.(A) Mutiple tissue northern blots (Clontech) wereprobed with 1×106 cpm/ml of a [α-32P]dCTP-labeled SmaI-XhoI fragment of hSu(fu) cDNA, blotswere washed to a stringency of 0.2×SSC at 65°C,and exposed to film for 3 days. Each lane containsapproximately 2 µg of poly(A)+ RNA. sk. muscle,skeletal muscle; sm. intest, small intestine; pbl,peripheral blood leukocyte. (B) PCR analysis ofrelative hSu(fu) mRNA isoform expression levels invarious tissues. 50 ng of DNA from human cDNAexpression libraries was used as template to amplifyfragments of 679 and 828 base pairs, of hSu(fu)433and hSu(fu)484, respectively. A small amount of[33P]dATP was included in the reaction mix; after 30cycles, 10 µl were electrophoresed per lane on a 4%TBE acrylamide gel, which was dried and exposedto X-ray film for 4 hours.

4441Human Su(fu) negatively regulates Gli

mGli2, pRK-hGli3, pRK-mSlimb, pRK-hSu(fu)433 and SP6-Luciferase controlplasmid were transcribed and translated invitro using the TNT-coupled reticulocytelysate system (Promega), with 20 µCi[35S]methionine (Amersham) and SP6 RNApolymerase in a 50 µl reaction volume. 1 µl ofeach reaction was subjected to denaturingSDS-PAGE for approximate proteinquantitation. Equivalent amounts of eachprotein were used in binding assays.

Luciferase reporter assayThe reporter assay was performed inC3H10T1/2 cells as described (Murone et al.,1999), using a Dual-Luciferase ReporterAssay System (Promega, Inc). Differences intransfection efficiency were corrected bynormalizing the activity of the fireflyLuciferase reporter to the activity of acotransfected Renilla Luciferase internalcontrol.

RESULTS

Isolation and characterization ofhSu(fu) cDNAA BLAST search of the GenBank databaserevealed a mouse EST whose conceptualtranslation matched 55/91 amino acids ofthe dSu(fu) protein. To obtain the humanSu(fu) cDNA, we screened human fetallung and testis libraries with a [γ-32P]dCTP-labeled oligonucleotide probebased on the mouse EST sequence. Thelongest clone contained 1839 base pairs(bp), comprising 146 nucleotides of

Fig. 4. Distribution of Su(fu) mRNA inembryonic and adult rodent tissues.(A) Dorsal view and (B) side view of in situhybridization using a mouse Su(fu) probe towhole-mount embryonic day 8.5 (E8.5)mouse. (C-J) In situ hybridization of Su(fu) tosagittal sections (C,D,I,J) or coronal sections(E-H) of rat whole embryo (C,D), neural tube(E,F), or brain (G,H,I,J), at the indicated ages.J shows a higher power view of cerebellumshown in I. ps, primitive streak; np; neuralplate; hb, hindbrain; mb, midbrain; fb,forebrain; mes, mesoderm; som, somites; all,allantois; man, mandibular component of firstaortic arch; sc, spinal cord; ctx, cortex; di,diencephalon; cer, cerebellum; ton, tongue;eso, esophagus; liv, liver; gt, genital tubercle;lu, lung; dis, intervertebral disc; mg, midgut;nt, neural tube; epen, ependyma; nn,neocortical neuroepithelium; hip,hippocampus; ssz, striatal subventricularzone; th, thalamus; cau, caudate; hyp,hypothalamus; olf, olfactory bulb; ic, inferiorcolliculus; suc, superior colliculus. Scale bar,0.27 mm (A,B), 0.5 mm (C); 1.67 mm (D),0.16 mm (E); 0.59 mm (F); 1.14 mm (G);5.33 mm (H); 10 mm (I); 1.03 mm (J).

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upstream sequence, a 1299-bp ORF, and a 394-nucleotide3′UTR, in addition to an extensive poly(A) tail. Conceptualtranslation predicted an approximate 48-kDa protein containing433 amino acids (referred to as hSu(fu)433). A second clone,1720 bp in length, was identical to hSu(fu)433 from bases 313-1296 of the ORF (A in intiator ATG = base 1), then divergedcompletely at the 3′ end. This clone, which was missing the 5′UTR and starting methionine, comprised a partial 1139-bp ORF,in addition to a 581-bp 3′-UTR and a poly(A) tail. Assumingidentity to hSu(fu)433 at the 5′ end, the second cDNA predicteda protein identical to hSu(fu)433 with an additional 52 aminoacids at the carboxy terminus (referred to as hSu(fu)484).Alignment of hSu(fu) with dSu(fu) revealed a 37% identity atthe amino acid level (Fig. 1), which increased to 63% allowingconservative amino acid substitutions. During preparation of thismanuscript, a mouse Su(fu) cDNA was deposited in GenBank(accession no. AJ131692), and is included in the sequencealignment. The longer splice variant of human Su(fu) exhibits97% sequence identity to the mouse Su(fu). A search of hSu(fu)against the Prosite database revealed 15 potentialphosphorylation sites (16 in hSu(fu)484), several of which wereconserved between Drosophila and vertebrate Su(fu) (indicatedin Fig. 1). Three candidate PKA phosphorylation sites wereidentified in hSu(fu) and none in dSu(fu). However, by includingin our search strategy several less active potential PKAphophorylation site motifs, we identified two additional sites inhSu(fu) and five such sites in dSu(fu) (Fig. 1). The PESTalgorithm (Rechsteiner and Rogers, 1996) identified a highscoring PEST sequence (score=26.29), which spanned aminoacids 280-289, and was conserved between human and mouse.The hSu(fu) gene was mapped to chromosome 10, region q24-q25 by FISH analysis (Fig. 2).

Northern blot analysis and semi-quantitative PCRHigh stringency hybridization to human poly(A)+ RNArevealed hSu(fu) mRNA to be expressed widely in bothembryonic and adult tissues (Fig. 3A). A major transcript ofapprox. 5.5 kb was detected in all tissues examined, predictinga relatively long 5′ UTR for the hSu(fu) message that wasabsent in our isolated clones. In several tissues, a 2.5 kbtranscript could also be seen, and in testis two smallertranscripts of approx. 2.0 and 1.0 kb were also detectable.Semi-quantitative PCR was performed on cDNA derived froma number of human tissues, to determine the relative expressionlevels of the two alternatively spliced hSu(fu) mRNAtranscripts. Both transcripts could be detected in all tissuesexamined; however, hSu(fu)484 was always the more abundantisoform (Fig. 3B). Adult testis contained the highest relativelevel of the shorter isoform, in which the ratio ofhSu(fu)433:hSu(fu)484 was approximately 1:3 (Fig. 3B).

In situ hybridization to hSu(fu) mRNAWhole-mount in situ hybridization revealed Su(fu) mRNA tobe widely expressed in E8.5 mouse (Fig. 4A,B), the earliestdevelopmental time point examined. Labeling was uniformlyintense throughout the developing neural plate. Only the anlageof the heart was not specifically labeled at this stage (Fig. 4B).In E11.5 rat, Su(fu) message remained widespread throughoutthe central nervous system, spinal cord and somites (Fig. 4C).In E11.5 and E15.5 rat spinal cord, a prominent signal was seenwithin the developing neuroepithelium of the ventricular zone

(Fig. 4E,F), a region of active cellular proliferation. Tissuesthroughout the E15.5 embryo, including the brain, spinal cord,gut, lung and testis were labeled for Su(fu); the liver displayedonly a very low signal (Fig. 4D). In the P1 rat brain, Su(fu)mRNA was widely expressed, with prominent signalsoverlying the neuroepithelium, subventricular zone andhippocampal neuronal cell fields (Fig. 4G). Message wasprofoundly downregulated in adult brain yet still weaklydetectable throughout; relatively high expression was observedin the hippocampus, cerebellar granule and Purkinje cell layers,and olfactory bulb (Fig. 4H-J).

In a cross section of adult mouse testis, Su(fu) mRNA wasintensely expressed in a subset of seminiferous tubules,suggesting that its transcription may be regulated according tothe stages of germinal cell differentiation (Fig. 5A). Su(fu)message was observed as a ring of silver grains over the regionof developing spermatocytes (Fig. 5C); in many sites within thetubule, highest expression was concentrated in the center, wherethe latest stages of germinal cell differentiation occur (Fig. 5D).Hybridization of a sense strand control probe to an adjacenttissue section showed no signal above background (Fig. 5B).

Immunocytochemistry, biochemical interactions andbiological activities of hSu(fu)Since previous studies demonstrated binding of dSu(fu) to Ci(Monnier et al., 1998), the Drosophila Gli homologue, we askedwhether a similar interaction might exist between their humanprotein counterparts. We used immunocytochemistry tovisualize the subcellular localization of transientlyoverexpressed hSu(Fu) and hGli in transfected C3H10T1/2 cells.When individually expressed, hSu(fu) and hGli were extensivelydistributed throughout the cytoplasm; hGli was sometimesdetected in the nucleus (data not shown). Double labeling ofcotransfected C3H10T1/2 cells revealed an extensive overlap inthe two staining patterns (Fig. 6, row A). Moreover, hGlifrequently appeared to be depleted from the cytoplasm andinstead concentrated in widely dispersed punctate, denselystained regions, which also labeled strongly for hSu(fu) (Fig. 6,row A). These densely stained regions, which were not seen incells overexpressing hSu(fu) alone, and always stained for bothproteins, might represent cytoplasmic sequestration of hGli byhSu(fu). Double labeling of cotransfected cells with an anti-β-tubulin antibody showed hSu(fu) to be partially colocalized withtubulin, predominantly in perinuclear regions (Fig. 6, row B),although Su(fu) was also seen in areas devoid of tubulin staining.Interestingly, treatment of cotransfected cells with nonionicdetergent prior to fixation resulted in frequent nuclearlocalization of hSu(fu) antigen (data not shown). In COS-7 cells,hGli was predominantly nuclear when expressed either aloneor with hSu(fu)433, yet relocalized to the cytoplasm whencoexpressed with hSu(fu)484 (Fig. 6C).

To determine whether hSu(fu) and hGli formed a physicalcomplex, we looked for coimmunoprecipitation of the twoproteins from transiently transfected 293 cells. We found thathSu(fu) and hGli could be readily coimmunoprecipitated fromcells expressing both proteins (Fig. 7A). This interactionappeared not to involve indirect protein-DNA associations,since inclusion of ethidium bromide in the immunoprecipitationreaction (Lai et al., 1992) did not inhibit complex formation(Fig. 7A). The hSu(fu)-hGli interaction was confirmed using anin vitro binding assay. For this purpose, bacterially produced

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4443Human Su(fu) negatively regulates Gli

GST-hSu(fu) protein was loaded on glutathione-sepharosebeads and examined for its ability to retain in vitro-translated35S-labeled hGli. We found that hGli was specifically retainedon GST-hSu(fu) glutathione-sepharose beads, but not on beadsloaded with GST alone (Fig. 7B).

In further experiments, we examined the ability of the Glihomologues Gli2 and Gli3 to interact with hSu(fu). Both 35S-labeled mGli2 and hGli3 but not Luciferase, a negative control,were specifically retained by GST-hSu(fu)-conjugated beads(Fig. 7B). A version of hGli3 in which the 9E10 c-myc epitopewas fused to the extreme carboxy terminus of the protein didnot bind to hSu(fu) in this assay (data not shown), indicatingthat the carboxy-terminal region of Gli3 is important for theinteraction. Interestingly, 35S-labeled Su(fu) also bound toGST-hSu(fu) (Fig. 7B), and hSu(fu) was found to bind to itselfin a 2-hybrid assay (data not shown), indicating that hSu(fu)may function as a dimer.

Our binding data suggested that the activity of vertebrate Glismay be regulated by interaction with Su(fu). Thus, we examineddirectly whether hSu(fu) could modulate the activity of hGli ina functional Gli reporter assay. To this end, nine copies of a Glibinding site (Sasaki et al., 1997) were linked to a Herpessimplex virus Thymidine kinase minimal promoter, whichdirects the transcription of a reporter firefly Luciferase gene.Expression of Luciferase from this construct was shown to bespecifically regulated by Gli and by components of the Shhreceptor (Murone et al., 1999). As previously demonstrated(Murone et al., 1999), cotransfection of C3H10T1/2 cells withthe Luciferase reporter construct and either empty vector or anexpression plasmid encoding an irrelevant protein (pRK-GFP)resulted in very low levels of Luciferase activity (Figs 7C, 8).In contrast, cotransfection of the reporter gene with an hGliexpression plasmid resulted in an approximately 40- to 70-foldincrease in the level of Luciferase activity (Figs 7C, 8).Consistent with the notion that dSu(fu) is a negative regulatorof Ci, hGli-mediated reporter activation was significantlysuppressed in the presence of coexpressed hSu(fu)433 orhSu(fu)484, but not an irrelevant protein (Fig. 7C). Thus, ourfindings suggest that the physical interaction of hSu(fu) withhGli leads to inhibition of its transactivator function.

Su(fu) might repress Gli activity in part by altering itsprocessing or degradation, or by influencing its DNA-bindingactivity. To begin to examine these possibilities, we looked forinteractions between hSu(fu) and a vertebrate homologue ofSlimb/βTrCP, an F-box containing protein implicated intargeting of Ci and other proteins to the Ubiquitin-proteasomaldegradation pathway (Jiang and Struhl, 1998; Margottin et al.,1998; Yaron et al., 1998). We found that in vitro-translated 35S-labeled Slimb indeed specifically bound to Su(fu) in the GSTpull-down assay (Fig. 7B). To investigate a potential functionalrole for this interaction, we tested the effect of Slimb on Glitranscriptional activity. In our functional Gli reporter assay,Slimb alone did not alter Gli-induced reporter expression;however, when cotransfected with hSu(fu), Slimb significantlypotentiated the inhibitory effect of Su(fu) on Gli activity (Fig.8). This potentiation was not seen with substitution of Slimb∆F,a truncated version of Slimb lacking the F-box domain (Fig. 8).

DISCUSSION

We have identified two isoforms of a human protein exhibiting

63% similarity to dSu(fu), and a developmental expressionprofile consistent with a role in vertebrate HH signaling. Theidentification of multiple hSu(fu) isoforms suggests that thegene is subject to alternative splicing, and that the divergentcarboxy terminus of the protein may serve an importantregulatory function. While minor differences were observed inthe extent of Gli repression mediated by the two proteins in aGli reporter assay, further studies are required to determine thefunctional role of alternative splicing in the regulation of HHsignaling in vivo. In preliminary studies, we found that the ratioof the two mRNA transcripts differed between tissues, withhSu(fu)484 predominating.

Immediately prior to submission of this manuscript, a mouseSu(fu) cDNA was deposited in GenBank. Alignment of thelonger human splice variant with mouse Su(fu), revealed a 97%sequence identy at the amino acid level. Several potentialphosphorylation sites conserved between hSu(fu) and dSu(fu)were identified, two of which are candidate PKAphophorylation sites; the only other protein motif identifiedwas a high-scoring PEST domain (Rechsteiner and Rogers,1996) in the carboxy terminal half. By FISH analysis, wemapped the hSu(fu) gene to chromosome 10q24-25.Interestingly, two loci for tumor suppressor genes have beenproposed within the interval 10q.23-qter, based on loss ofheterozygosity (LOH) analysis in a number of tumors,including glioblastoma multiforme, prostate cancer, malignantmelanoma and endometrial cancer (Albarosa et al., 1996; Grayet al., 1995; Peiffer-Schneider et al., 1998; Rasheed et al.,1995). Two other candidate tumor suppressor genes foundmutated in a number of cancers have also recently beenmapped to this region: MMAC1/PTEN at 10q23.3 (Li et al.,1997; Steck et al., 1997) and DMBT1 (deleted in malignantbrain tumors) at 10q25.3-26.1 (Mollenhauer et al., 1998).

By northern analysis, hSu(fu) was found to be widelyexpressed in both fetal and adult tissues. The presence ofhSu(fu) mRNA in many adult tissues suggests that they maycontinue to posess a functional HH signaling pathway, and thathSu(fu) might be required therein to suppress Gli activity.Conceivably, loss of hSu(fu) function in these tissues couldprovide an additional route to activation of HH signaling, andconsequent oncogenesis. In light of this possibility, themapping of hSu(fu) to a known tumor suppressor locus isparticularly intriguing.

Examination of the developmental expression of rodent Su(fu)by in situ hybridization revealed that many HH-responsivetissues prominantly expressed Su(fu) mRNA (see Fig. 4D),including Shh-responsive embryonic neural folds and neuraltube (Hammerschmidt et al., 1997; Ingham, 1995), presomiticmesoderm and somites (Fan et al., 1995), and embryonic foregut,esophagus and lung (Litingtung et al., 1998; Motoyama et al.,1998), Ihh-responsive cartilage (Vortkamp et al., 1996), andDhh-responsive testis (Bitgood et al., 1996). Expression wasmaintained in a subset of cells within the adult brain, includinghippocampal pyramidal and granule cells, cerebellar granule andPurkinje cells, and olfactory bulb granule cells, suggesting thatregions which remain mitotically active or retain the capacity forsuch activity may require the continued expression of Su(fu). Inadult rat cerebellum, expression of Su(fu) overlapped with thatof Shh, Smo and Patched mRNA in Purkinje cells (Fig. 4J)(Traiffort et al., 1998). In humans, Shh signaling is known toplay an important role in cerebellar development (Wechsler-

4444

Reya and Scott, 1999). This is evidenced by theinvolvement of Patched mutations in the etiology ofNaevoid basal cell carcinoma (Gorlin) syndrome (Hahnet al., 1996; Johnson et al., 1996), which is associatedwith an increased frequency of cerebellarmedulloblastoma. Also, mice heterozygous for atargeted disruption of the Patched gene developmedulloblastomas (Goodrich et al., 1997). The presenceof Shh signaling components in adult cerebellum mayindicate a continued role for this pathway in cerebellarmaintenance.

In testis, expression of Su(fu) mRNA in developinggerm cells overlapped with that of Patched2, a secondvertebrate HH-binding protein with homology toPatched, and a vertebrate Fu homologue (Carpenter,1998). Additionally, both Gli and Gli3 are expressed indeveloping spermatagonia (Persengiev et al., 1997);together, the data support the hypothesis that Sertoli cell-

D. M. Stone and others

Fig. 5. Tissue distribution of Su(fu) in adult mouse testis.(A) Cross section of adult testis hybridized to Su(fu) probe.Higher magnification views (C-D) demonstrate Su(fu)mRNA localization to developing spermatocytes (C) or, insome regions, to the center of seminiferous tubules (D)where the latest stages of germinal cell differentiation occur.(B) Hybridization of the testis with a sense strand controlprobe. st, seminiferous tubule; ta, tunica albuginea; sg,spermatogonia; sc, spermatocytes; lc, leydig cells; sm,mature sperm; lu, lumen. Scale bar, 1.0 mm (A,B) and 0.065mm (C-D).

Fig. 6. Subcellular localization of hSu(fu) andhGli. (A) Colocalization of hSu(fu) and hGli intransfected CH310T1/2 cells. Cells werecotransfected with pRK-hSu(fu)433 and pRK-hGli, and proteins were immunocytochemicallystained 24 hours later and visualized byfluorescence microscopy. Transfected cells werefixed, permeabilized and double-labeled for bothhGli and hSu(fu), using anti-c-myc and anti-hSu(fu) primary antibodies followed by cy2-conjugated anti-mouse IgG and cy3-conjugatedanti-rabbit IgG, respectively. A singlemicroscope field is shown, using filters for cy2(left panel), cy3 (center panel), or an overlap ofthe two images (right panel) in which yellowindicates colocalization. Note the differentstaining patterns observed in two transfectedcells: one shows uniform cytoplasmic staining,the other shows punctate labeling. (B) Partialcolocalization of hSu(fu) with β-tubulin inCH310T1/2 cells. Cells were transfected withhSu(fu)433 and hGli, then double-labeled 24hours later for β-tubulin and Su(fu) using anti-tubulin and anti-hSu(fu) antibodies, followed bycy2-anti-mouse and cy3-anti-rabbit IgGs. Asingle field is shown as visualized byfluorescence microscopy using filters for cy2(left panel), cy3 (center panel) or an overlap ofthe two images (right panel). (C) Subcellularlocalization of hGli in COS-7 cells whentransfected alone (left panel), or together withhSu(fu)484 (middle panel) or hSu(fu)433 (right panel). Insets show double-labeling for hSu(fu) in indicated cells. Bar, 20 µm.

4445Human Su(fu) negatively regulates Gli

derived Dhh (Bitgood et al., 1996) may signal developing germcells through a Smo-Patched2 receptor complex. Su(fu) was notobserved in the interstitial Leydig cells, the site of Patched geneexpression in adult testis (Bitgood et al., 1996; Carpenter, 1998).The presence of Su(fu) mRNA in tissues responsive to Shh, Ihhand Dhh suggests that the same signaling components andmechanisms may be used by all mammalian HH familymembers.

Consistent with a role for Su(fu) in vertebrate HH signaling,immunocytochemical localization of coexpressed hSu(fu) andhGli in cultured cells revealed the two proteins to becolocalized (Fig. 6A,C). While the staining pattern observedfor either of the splice variants of hSu(fu) appeared similarwhen expressed alone, the shorter isoform was more

prominently localized to punctate cytoplasmicdensities upon coexpression with hGli (data notshown). Interestingly, selective extraction of cellswith nonionic detergent prior to fixation revealednuclear localization of hSu(fu) labeling (data notshown). This effect might be due to anunmasking of hidden antigenic sites, or anenhancement of the relative nuclear labelingintensity due to elimination of backgroundcytoplasmic staining. Nuclear hSu(fu)localization was not altered by pretreatment of

cells with recombinant soluble N-Shh, and was often, althoughnot always, coincident with nuclear hGli labeling (see Fig. 6C).Although the hSu(fu) sequence does not contain a conventionalnuclear localization signal, Reinhardt’s method fornuclear/cytoplasmic discrimination (Reinhardt and Hubbard,1998) predicts hSu(fu) to be predominantly nuclear.

In Drosophila, Ci is found in complex with Su(fu), Fu(Monnier et al., 1998), and Costal2 (Robbins et al., 1997;Sisson et al., 1997), a kinesin-related microtubule-associatedprotein thought to repress Hh signal transduction by tetheringthe signaling machinery to the cytoskeleton. We thus examinedwhether hSu(fu) and hGli colocalized with microtubules inmammalian cells. Labeling of transfected cells with an anti-β-tubulin antibody revealed a partial overlap in the staining

Fig. 7. (A) Coimmunoprecipitation of hGli and hSu(fu)in transiently transfected 293 cells. Cells weretransfected with expression plasmids encodingindicated proteins (4 µg each, normalized to a total of 8µg with empty pRK vector). Cells were lysed 24 hourslater, and the lysate immunoprecipitated with anti-flagM2 (for flag-tagged hSu(fu)) or anti-c-myc (for myc-tagged hGli) antibodies. Ethidium bromide (EtBr) wasadded to some lysates (as indicated), to preclude DNA-dependent protein associations. Protein complexeswere subject to denaturing SDS-PAGE on 4-12%NuPAGE gels, transferred to nitrocellulose, and probedwith anti-myc or anti-flag antibodies, as indicated.Antibodies were visualized by ECL detection.(B) GST-fusion protein binding assay. Proteins werelabeled with [35S]methionine by in vitro transcription-translation, and incubated with glutathione-sepharosebeads conjugated to either GST-hSu(fu) or GST, for 2hours at 4°C. After washing, bound proteins wereeluted by boiling in SDS loading buffer, and sampleswere subjected to 10% or 8% (hSu(fu) only) denaturingSDS-PAGE. Gels were fixed, amplified, dried andexposed to film. The amount of labeled protein used ineach reaction was four times that shown in the input(in) lane. Lucif, Luciferase. (C) Gli activation reporterassay. C3H10T1/2 cells in 6-well plates weretransiently transfected with a Gli-binding siteLuciferase reporter plasmid (1 µg) together withexpression constructs for hGli, hSu(fu)433, hSu(fu)484or empty vector (pRK) (0.25 µg each in left panel),alone or in combination. The total amount of effectorplasmid was normalized to 1 µg with pRK-GFP. Therelative Luciferase activity in cell lysates was measured24 hours after transfection and was normalized toRenilla Luciferase activity (pRL-TK; 0.0025 µg/well).Values are the means ± s.d. of duplicate determinationsof duplicate transfections from a representativeexperiment out of three.

4446

patterns of β-tubulin and either hSu(fu) or hGli (Fig. 6B anddata not shown), allowing the possibility that, as in fly, theactivity of HH signaling components might be regulated byassociation with cytoskeletal elements.

A physical association between hGli and hSu(fu) wasdemonstrated in two different assay systems. First, either splicevariant of hSu(fu) could be co-immunoprecipitated with hGlifrom cotransfected 293 cells, using antibodies to epitope tagson hSu(fu) and hGli (Fig. 7A). Second, 35S-labeled in vitro-translated hGli bound specifically to a GST-hSu(fu) fusionprotein in an in vitro binding assay (Fig. 7B). Our resultscomplement those of Monnier and collegues (1998) whodemonstrated an analogous interaction between Drosophila Ciand dSu(fu). These authors found that Su(fu) could act as a linkbetween Fu and Ci in the formation of a trimolecular complex(Monnier et al., 1998). They predicted that activation of Fumight trigger the dissociation of dSu(fu) and Ci, conceivablythrough phosphorylation of the PEST sequence in dSu(fu) andconsequent dSu(fu) degradation. Consistent with thishypothesis, we have observed a decrease in the amount ofhSu(fu) co-immunoprecipitated with hGli, upon exposure ofcotransfected C3H10T1/2 cells to soluble N-Shh (M. Muroneand F. J. de Sauvage, unpublished).

We further examined the ability of Gli2 and Gli3, twoadditional members of the Gli family of zinc finger transcriptionfactors, to interact with hSu(fu). The three Gli proteins appearto subserve both specific and redundant functions in HH-mediated developmental processes. This is indicated by theirdifferential expression patterns (Hui et al., 1994) and by the

observed phenotypes of mice harboring targeted disruptions inGli2 and Gli3 genes (Ding et al., 1998; Matise et al., 1998; Moet al., 1997; Motoyama, 1998). Both mGli2 and hGli3 werefound to bind specifically to GST-hSu(fu) protein in our in vitrobinding assay (Fig. 7B). Our data thus support a role for hSu(fu)in regulating the activity of all members of the Gli protein family.

Previous genetic studies have suggested that the interactionof dSu(fu) with Ci may inhibit Ci by preventing its maturationinto a transcriptionally active form (Monnier et al., 1998;Ohlmeyer and Kalderon, 1998). This inhibition, is thought tobe relieved by reception of the Hh signal. We directly examinedwhether hSu(fu) could influence the activity of hGli in a Glitranscriptional activation reporter assay (Murone et al., 1999).We found that hGli could activate reporter expression up to 70-fold, and this activation was dramatically suppressed bycoexpression of hSu(fu) (Fig. 7C). The finding that Su(fu) didnot completely abolish Gli-mediated activation might reflectdifferences in relative protein levels due to unequal transfectionor expression efficiencies. In this regard, evidence derivedfrom Drosophila genetics (Ohlmeyer and Kalderon, 1998)demonstrates the critical role of the stoichiometric ratiobetween Ci and dSu(fu) in determining cellular response.

In addition to its interaction with Gli family members, hSu(fu)associated with itself and with a vertebrate homologue of Slimb(Fig. 7B). The former interaction was independently identified ina yeast 2-hybrid screen, in which Su(fu) from a human testislibrary was isolated as an interacting partner with full-lengthhSu(fu). Homo- or heterodimers of hSu(fu) might function tobring together other effector proteins, thus allowing fordifferential regulation of Gli activity. The association of Su(fu)with Slimb prompted us to investigate a potential role for thisinteraction in Gli regulation. Genetic data from Drosophilaimplicate Slimb as a negative regulator of Hh signaling (Jiangand Struhl, 1998; Theodosiou et al., 1998), by enhancing Cidegradation. Slimb contains an F-box and several WD-40 repeatdomains which function, respectively, as a binding site forcomponents of the E2 ubiquitin-conjugating protein degradationcomplex (Bai et al., 1996; Margottin et al., 1998; Skowyra et al.,1997) and as protein-protein interaction regions (Neer et al.,1994). When tested in a functional Gli activity assay, Slimb alonehad no effect on Gli-mediated transactivation, yet it potentatedthe inhibitory effect of Su(fu) on Gli activity (Fig. 8). The Slimb-enhanced suppression of Gli required the presence of the F-boxdomain, consistent with involvement of the multimeric SCF E3ubiquitin ligase (Skowyra et al., 1997). In related experiments,we found that Slimb and hSu(fu) (either isoform), as well asSlimb and hGli, could be co-immunoprecipitated from 293 cellsoverexpressing the two proteins (data not shown). The datasupport a model in which Su(fu) may act as an adapter proteinto link Gli to the Slimb-SCF-dependent degradation pathway(Skowyra et al., 1997). A similar function was demonstrated forthe HIV-1 protein Vpu in the association between βTrCP, analternatively spliced isoform of human Slimb (Theodosiou et al.,1998), and CD4 (Margottin et al., 1998). However, the preciserole of Slimb in this context remains to be defined. Unlike Ci, Gliappears not to undergo proteasome-dependent cleavage into atruncated repressor form (Yoon et al., 1998; Dai et al., 1999).Furthermore, recent studies in Drosophila cl-8 cells suggest thatthe formation of Ubiquitin-Ci conjugates – presumably mediatedby Slimb – is not under Hh regulation (Chen et al., 1999). Thus,we cannot exclude the possibility that the Slimb/Gli/Su(fu)

D. M. Stone and others

Fig. 8. Slimb enhances the negative effect of Su(fu) on Gli-mediatedtranscriptional activation. Gli-regulated Luciferase reporter assaywas performed as described above, using expression constructs forthe indicated proteins (0.5 µg for pRK-Slimb and pRK-Slimb∆F,0.25 µg for all other constructs; hSu(fu) represents hSu(fu)484). Therelative Luciferase activity in cell lysates was measured 24 hoursafter transfection and was normalized to Renilla Luciferase. Valuesare the mean ± s.d. of duplicate determinations of duplicatetransfections from a representative experiment out of two. Thevariation in the magnitude of the Su(fu)-mediated suppression of Gliactivity (compare to Fig. 7C) reflects differences in cell passagenumber and density at the time of assay, both of which affect reporterresponse. Therefore, although intra-assay variability is generallyquite low, direct comparisons cannot be made between assaysperformed on separate days. P<0.01 for Gli+Su(fu) versusGli+Su(fu)+Slimb, by one-way ANOVA.

GFPSu(

fu)

Slimb

Slimb

F Gli

Gli+Slim

b

Gli+Slim

b F

Gli+Su(

fu)+

Slimb

Gli+Su(

fu)+

Slimb

F

Gli+Su(

fu)

∆∆∆

Rel

ativ

e lu

cife

rase

act

ivity

60

40

20

0

4447Human Su(fu) negatively regulates Gli

interaction functions, in part, to maintain the steady state level ofGli and is not directly involved in HH signaling.

Our results suggest that Gli may be regulated by Su(fu) atmultiple levels. Alterations in Gli protein stability by recruitmentof the Ubiquitin degradation machinery, represents one potentialregulatory step. However, preliminary studies suggest that,although both forms of Su(fu) inhibit Gli activity, they exertdifferent effects on Gli stability/solubility and subcellularlocalization. Thus, by analogy to Drosophila systems (Ohlmeyerand Kalderon, 1998; Chen et al., 1999), the predominant role ofSu(fu) may be to sequester Gli, and, through this association,prevent its post-translational modification into a transcriptionallyactive form.

In sum, the biochemical interactions which we havedemonstrated between hSu(fu) and Gli family members, inconjunction with results from a Gli transactivation assay,complement genetic studies in Drosophila and support the ideathat Su(fu) is a direct negative regulator of Gli. This regulationmay occur at multiple levels, affecting the stability, processingand cellular localization of Gli. Our data further emphasize theimportance of the relative intracellular concentrations ofdifferent signaling components in determining the cellularresponse to HH family members, and extend the conservation(with some notable differences) of Hh signaling componentsand mechanisms from Drosophila to human.

Note added in proof We have recently identified an ~5 kb hSu(fu) cDNA whichcontains a 2715-bp extension of the hSu(fu)484 cDNA in the3′ UTR. This cDNA correlates in size with the most abundantmRNA transcript on northern blots, and likely represents useof an alternative polyadenylation signal. Additionally, we haveidentified a third alternative splice variant of hSu(fu) (referredto as hSu(fu)481) encoding a protein identical to hSu(fu)433up to residue 431, and including the following additional 50amino acids at the carboxy terminus:

HVRWPFFFSLLPFIDFLAHPSSSPLAALDGTPSWGAGHE-CLMDSGPGACV.

While this paper was in press, three groups haveindependently described the cloning of mammalian Su(fu) andits ability to bind Gli family members (Ding et al. (1999) Curr.Biol. 9, 1119-1122; Kogerman et al. (1999) Nature Cell Biol.1, 312-319; Pearse et al. (1999) Dev. Biol. 212, 323-336). Inagreement with two of these reports, we find that hSu(fu)484sequesters Gli in the cytoplasm of cotransfected COS-7, NIH-3T3, or C3H10T1/2 cells. However, neither splice varianthSu(fu)433 or hSu(fu)481 similarly alters Gli subcellulardistribution, although both can interact with, and suppress theactivity of Gli.

The GenBank accession numbers for hSu(fu) are AF144231 andAF159447.

We thank Kenneth Kinzler for the Gli cDNA, Mike Ruppert for theGli3 cDNA, the Genentech DNA synthesis group for oligonucleotidesynthesis and purification, and Wei-Hsein Ho for epitope-taggedhSu(fu). We are grateful to SeeDNA Biotech (Toronto, Ontario,Canada) for performing the FISH analysis, to Greg Bennett for helpwith antibody production, to Christa Gray and Alan Zhong for DNAsequencing, and to Wayne Anstine and Evelyn Berry for assistancewith graphics and manuscript preparation.

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