Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.


Introduction
Hedgehog (Hh) signaling regulates numerous developmental processes and is implicated in multiple cancers, wound healing and pain sensation in adults [1][2][3]. The Hh ligand acts as a morphogen to induce differential cell responses based on distinct activity thresholds of its signaling transduction cascade [4][5][6]. Mis-regulation of Hh signaling affects cell specification and proliferation during development and causes several types of cancer such as glioblastoma or basal cell carcinoma [7,8]. In the absence of Hh, the receptor Patched (Ptc) inhibits the G-protein coupled receptor Smoothened (Smo) [9]. Inhibition of Smo promotes the assembly of an antagonistic molecular complex composed of Costal 2 (Cos2), a kinesin-related motor protein, Cubitus interruptus (Ci), the key transcriptional effector of Hh [10,11], and several protein kinases [12]. This complex phosphorylates the full length, transcriptionally active form of Ci, Ci 155 . Phosphorylated Ci 155 is ubiquitinated by a SCF (Skp1-Cullin1(Cul1)-F-box) E3 ligase complex [13] and partially cleaved to generate a transcriptional repressor form, Ci 75 , which leads to the transcriptional silencing of Hh target genes [14,15]. The Hh signaling cascade is activated by the binding of Hh to Ptc and Ihog (Interference hedgehog) [16], resulting in the release of Smo inhibition. Activated Smo can interact physically with Cos2 [17][18][19][20]. This interaction prevents the formation of the Hh signaling antagonistic complex and cleavage of Ci 155 . As a result, levels of Ci 155 increase in the cytoplasm, promoting its translocation to the nucleus and the transcription of downstream target genes such as decapentaplegic (dpp) or ptc (Fig 1A).
Previous studies have shown that Cos2 is a key modulator of Hh signaling, and that it facilitates kinase-mediated phosphorylation of Ci and promotes partial degradation of Ci [21]. Loss of Cos2 leads to ectopic activation of Hh signaling and pattern duplications in the Drosophila wing [11], whereas over-expression of Cos2 inhibits Hh signaling [22], suggesting that Cos2 is both necessary and sufficient for Hh signaling. In vertebrates, the core components of Hh signaling are conserved, including Cos2. Cos2 has two vertebrate orthologs, Kif7 and Kif27 [23,24]. Kif7 has been proposed to function similarly to Cos2, because Kif7 knockout mice and zebrafish mutants show an up-regulation of Sonic Hedgehog (Shh) signaling [25][26][27]. In addition, Kif7 can interact physically and modulate the activity of the GLI transcription factors, the mammalian homologs of Ci [27,28]. Moreover, Cos2 can functionally replace Kif7 [27], demonstrating a molecular conservation between vertebrate and invertebrate homologues. In humans, patients carrying KIF7 allelic variants display a spectrum of phenotypic severity ranging from hydrolethalus or Acrocallosal syndromes to Meckel and Joubert syndromes [28,29]. Hence, proper function of Kif7 activity is essential for correct Hh signal transduction and is likely to be regulated tightly. Previous studies have shown that Cos2 (Kif7) is phosphorylated by a kinase, Fused, which mediates the strength of differential Hedgehog signaling [30,31]. To date, however, no data support a role for ubiquitination in the regulation of Cos2.
We describe the identification and characterization of Ubr3, a novel regulator of Hh signaling. Ubr3 belongs to the UBR protein superfamily, characterized by a 70-residue zinc finger domain UBR box [37]. Recent studies showed that Ubr3 can polyubiquitinate target proteins [38] involved in multiple biological processes, including olfactory organ function in mice [39], denticle patterning in Drosophila [40], DNA damage repair in yeast [38], apoptosis in flies [41], homoeostasis in the heart [42], and breast cancer [43].
Here we show that Ubr3 promotes Hh signaling by mediating the ubiquitination and degradation of Cos2/Kif7. Loss of Ubr3 elevates the levels of Cos2, resulting in a decrease in Ci 155 and transcriptional silencing of Hh target genes. Loss of ubr3 in flies and zebrafish affects eye development, as well as neuronal specification and somite development in zebrafish. Ubr3 regulates the ubiquitination and degradation of Kif7 in mammalian cells, and transcription of the Shh target ptch2 is strongly decreased in the retina of ubr3 mutant zebrafish. Taken together, our data suggest that Ubr3 is an evolutionarily conserved, positive regulator of Hh signaling that regulates Cos2/Kif7 ubiquitination and degradation.

Loss of ubr3 results in Hh signaling defects in Drosophila
To identify novel components in developmental signaling pathways, we isolated mutations that affect eye and/or wing morphogenesis in a mosaic forward genetic screen of approximately 6000 X-linked lethal mutations in Drosophila [44][45][46][47][48]. We identified an essential complementation group ubr3, consisting of two alleles (ubr3 A and ubr3 B ). Both ubr3 A and ubr3 B hemizygous mutants die as 1 st instar larvae. Homozygous mutant clones of both alleles cause delayed differentiation of photoreceptors in the morphogenetic furrow of eye imaginal discs (Fig 1B and S1A  Fig). This is revealed by the delayed expression of Senseless, an R8 photoreceptor marker [49,50] and Elav (Embryonic lethal abnormal vision), a marker for photoreceptors [51]. Since delayed differentiation of photoreceptors is observed when Hh signaling is lost [52], we hypothesized that ubr3 mutations may impair Hh signaling. To assess the activation of Hh signaling in ubr3 mutant clones, we examined expression of a Hh reporter, dpp-lacZ [53] and the active form of Ci, Ci A . Both dpp-lacZ and Ci 155 are lost in ubr3 mutant clones in the morphogenetic furrow ( Fig 1C-1D' and S1B Fig). We and others also noticed an increase in apoptosis in ubr3 mutant cells [41]. To exclude the possibility that the Hh signaling defect in ubr3 mutant cells is due to apoptosis, we over-expressed the anti-apoptotic gene p35 in ubr3 clones. As shown in S1C Fig, the delayed differentiation of photoreceptors is not rescued although apoptosis is suppressed (S1D and S1E Fig). Hence, Hh signaling is impaired in ubr3 mutant cells.
Alleles of ubr3 map to a small deficiency that uncovers~11 genes including ubr3 (CG42593) and l(1)G0193 (S1F Fig). Both alleles (ubr3 A and ubr3 B ) fail to complement the lethality associated with a P-element insertion in ubr3 (S1F and S1H Fig) [54]. ubr3 B carries a Leu 788 >STOP and ubr3 A carries a Phe 949 >Leu in ubr3 (Fig 1E). No mutations were found in l(1)G0193. A genomic rescue construct rescued the lethality of both ubr3 alleles (S1F and S1H Fig), and over-expression of the ubr3 cDNA in ubr3 B mutant clones rescued the loss of Ci 155 expression in the morphogenetic furrow (S1G Fig). Together, these data show that ubr3 is required for Hh signaling.
Ubr3 is a conserved E3 ligase and is expressed in Drosophila eye discs ubr3 encodes a 2219 amino acid protein, the Drosophila homolog of the mammalian RINGtype E3 ubiquitin ligase n-recognin 3 (UBR3) gene ( Fig 1E). Most UBR superfamily member proteins, including UBR1, UBR2, UBR4 and UBR5, function in the N-end rule pathway, a ubiquitin-dependent system where E3 ligases recognize N terminal residues of their targets and degrade them [37]. However, UBR3 does not bind to known N-end rule substrates, suggesting a different molecular function of Ubr3 from N-end rule E3 ligases [55]. Ubr3 contains a UBR moiety, a RING domain and a C-terminal auto-inhibitory (AI) domain ( Fig 1E) [38,39]. All three domains are highly conserved among fly, mouse and human (S1I Fig), suggesting that the molecular function of Ubr3 may be conserved.
To determine the expression pattern and protein localization of Ubr3, we raised a polyclonal antibody against a region between UBR domain and RING domain of Ubr3 (see Materials and Methods). The Ubr3 antibody specifically recognized a single 250 kDa band on Western blots of protein extracts from larval eye-brain complexes (Fig 2A). This band became more intense when a Ubr3 transgene was expressed (Fig 2A). Furthermore, immunofluorescent labeling of eye imaginal discs with our Ubr3 antibody revealed that the signal was severely diminished or lost within ubr3 B mutant clones (Fig 2B). Ubr3 is cytosolic and broadly expressed ( Fig 2C) and is enriched in the morphogenetic furrow of developing eye discs (Fig 2B), where Ci 155 and dpp-lacZ expression is elevated. The Ubr3 proteins in the cytosol are present in puncta that do not show obvious co-localization with a markers for different organelles (S2A- S2G Fig). These data suggest that elevated levels of Ubr3 positively correlate with the activation of Hh signaling.

Hh signaling activates the transcription of ubr3
To assess whether the enriched Ubr3 protein in the morphogenetic furrow ( Fig 2B) results from increased transcription of ubr3, we performed in situ hybridization experiments. As shown in Fig 2D, ubr3 was transcribed most abundantly in the morphogenetic furrow, in agreement with the protein enrichment shown in Fig 2B. Over-expression of ubr3 with an eyegone-Gal4 driver (eyg-Gal4; Fig 2E) expanded ubr3 expression domain in eye discs (Fig 2F), whereas ubr3 RNAi knockdown decreased expression of ubr3 in the center of the eye disc ( Fig  2G), showing the specificity of the RNA probes. We activated Hh signaling in the eyg positive cells by expressing a dominant-negative Ptc (ptc DN ) [56] or by down-regulating the expression of negative Hh regulators Cos2 or Cul1 by RNAi. In all cases, activation of Hh signaling elevated ubr3 mRNA levels in eye discs (Fig 2H-2J). In contrast, down-regulation of Ci by expressing Ci RNAi in the equator region of the morphogenetic furrow through eyg-Gal4 (arrow in S2H Fig) resulted in moderate loss of ubr3 transcription (white arrow in S2I Fig). Hence, Hh signaling positively regulates ubr3 expression at both the mRNA and protein levels. To assess whether different levels of Ubr3 proteins contribute in a dosage-dependent manner to Hh signaling, we manipulated the expression levels of Ubr3 in ubr3 B/B mutant cells by expressing a ubr3 cDNA at low or high levels. The actin-Gal4 driver used to express the ubr3 cDNA is temperature sensitive and leads to low expression at 18°C and medium to high expression at 25°C [57]. We then assessed Ci 155 expression in the mutant clones expressing discrete levels of Ubr3. Interestingly, when Ubr3 was expressed at low level at 18°C, Ci 155 expression was only partially restored (arrows in Fig 2K). However, high level of Ubr3 expression in ubr3 mutant cells fully rescued Ci 155 expression. In some cells, Ubr3 over-expression induced ectopic expression of Ci 155 posterior to the morphogenetic furrow (arrowheads in Fig 2L-2L"). In summary, these data suggest that Hh activation up-regulates transcription of ubr3, which in turn promotes Hh signaling.
Cos2 is up-regulated in ubr3 mutant cells  Fig 3A'), suggesting that Hh regulates the Ubr3-mediated down-regulation of Cos2.    Cos2 and Cul1 are both negative regulators of Hh signaling and loss of function of either gene causes ectopic activation of Hh signaling in eye discs [11,13,58]. Because both genes are up-regulated in cells lacking Ubr3, we tested whether over-expression of either gene is sufficient to phenocopy the ubr3 mutation. Over-expression of Cos2, but not Cul1, results in loss of Ci 155 in the morphogenetic furrow, similar to ubr3 mutants ( Fig 3B-3C'). Labeling with a Cos2 antibody showed that a subtle increase of Cos2 is sufficient to inhibit Ci 155 expression (S3D and S3D' Fig), implicating that Cos2 up-regulation in ubr3 mutant cells is relevant. Hence, up-regulation of Cos2, but not Cul1, is likely to be responsible for the Hh signaling defects observed in ubr3 mutants. This hypothesis is supported by the observation that reducing Cos2 protein levels in ubr3 mutant clones through cos2 RNAi restored Ci 155 levels and suppressed the morphogenetic furrow defects (arrows in Fig 3D and 3D' and S3E and S3E' Fig). In contrast, over-expression of cul1 RNAi in ubr3 mutant clones did not restore Ci 155 expression in the morphogenetic furrow (arrowheads in Fig 3E and 3E'), suggesting that Cul1 up-regulation was not the cause of Ci 155 loss. One likely reason why Ci 155 expression is not restored by Cul1 RNAi in ubr3 mutant clones in the morphogenetic furrow is that Cul1 RNAi does not completely remove Cul1 in ubr3 clones and the residual Cul1-Slimb E3 ligase activity may suffice to mediate processing of Ci 155 . Moreover, expression of ptc DN in ubr3 mutant clones did not rescue Ci 155 loss (arrowhead in Fig 3F and 3F'). These data show that loss of ubr3 causes a decrease in Hh signaling and a reduction in Ci 155 that can be restored by Cos2 down-regulation. Hence, ubr3 acts to attenuate the levels of Cos2, which enhances the activity of Hh signaling in the morphogenetic furrow.

Ubr3 possesses Ubiquitin E3 ligase activity and ubiquitinates itself
The RING domain of Ubr3 is not a canonical RING domain ( Fig 4A and 4A') [59]. To assess whether Ubr3 has E3 ligase activity, we performed an in vitro ubiquitination assay. Immunoprecipitation-purified Ubr3::GFP fusion proteins were incubated with E1 and E2 enzymes (HR6A) [39] and Flag-tagged Ubiquitin (Flag::Ub) peptides. Interestingly, Ubr3 poly-ubiquitinated itself, as shown in Fig 4B. Moreover, the UBR domain fragment may form a dimer when over-expressed, because a band of twice the molecular weight of GFP::UBR (~80 kDa) is detected ( Fig 4B). Co-immunoprecipitation assays with the over-expressed UBR domain indicated that it interacts with the Ubr3 full-length protein present in whole cell extracts of S2 cells ( Fig 4C). This suggests that Ubr3 interacts with the UBR domain of another Ubr3 molecule and that Ubr3 proteins poly-ubiquitinate each other.

Ubr3 binds to and poly-ubiquitinates Cos2
To test whether the up-regulation of Cos2 in ubr3 mutant cells is due to defective degradation by the proteasome, we performed a degradation assay of Cos2 in Drosophila S2 cells. We found degradation of Cos2 proteins begins 6 hours after treatment with a translational inhibitor cycloheximide (CHX) and that the level of Cos2 decreased to 10% after 10 hours of treatment ( Fig 5A). Addition of the proteasomal inhibitor MG132 suppressed the degradation of Cos2 (Fig 5A), suggesting that Cos2 proteins are degraded via the proteasome. The degradation of Cos2 is partially suppressed by down-regulation of Ubr3 by Ubr3 RNAi and promoted by overexpression of Ubr3 (Fig 5B), suggesting that Ubr3 mediates the degradation of Cos2.
Because ubiquitination is known to regulate protein abundance through proteasome-mediated degradation, Cos2 levels may be regulated via Ubr3-mediated ubiquitination. To determine whether Ubr3 interacts physically with Cos2 and to map which domains are required for this interaction, we performed co-immunoprecipitation assays. As shown in Fig  To exclude the possibility that Cos2 binds to Ubr3 indirectly via microtubules, we treated S2  Fig 5C), suggesting that Cos2 does not bind Ubr3 via microtubules. To identify which domain of Cos2 is critical for the interaction with Ubr3, we tested a series of deletion constructs of Cos2 ( Fig 5D) in co-IP assays with the UBR domain. We found that only the fragments bearing the N-terminal motor domain (MD) of Cos2 (Cos2ΔC1, ΔC2, and ΔC3) interacted with the UBR domain ( Fig 5E). Hence, Ubr3 binds to the N-terminal MD of Cos2 with its UBR domain.
To detect the ubiquitination of Cos2, we performed immunoprecipitation assays and examined the ubiquitination of Cos2 in S2 cells that express ubr3 ( Fig 4C). As shown in Fig  In addition, the HA signal exhibited a lower mobility shift compared to the major band detected by anti-Myc antibody (Fig 5F), indicating that these bands correspond to the ubiquitinated forms of Cos2. Over-expression of an E3 ligase dead form of Ubr3, in which the residues required for RING domain activity ( Fig 4A) were mutated to alanines, did not cause an increase in ubiquitination of Cos2 (Fig 5G, lane 1-3), suggesting that the E3 ligase activity of Ubr3 mediates the ubiquitination of Cos2. In addition, removing the Ubr3 binding domain of Cos2, Cos2ΔN1, abolished most of the ubiquitination of full length Cos2 (Fig 5G, lane 4-6). The residual ubiquitination of Cos2ΔN1 may result from endogenous full length Cos2 that coprecipitates with Cos2ΔN1 through dimerization [10,11].
To determine whether Ubr3 regulates Cos2 ubiquitination, we examined the levels of Cos2 ubiquitination when Ubr3 was either over-expressed or knocked down by RNAi. As shown in Fig 5F,

Hh signaling regulates Cos2 ubiquitination by Ubr3
We next tested whether Hh signaling regulates the ubiquitination of Cos2. Interestingly, we found that the ubiquitination of Cos2 was strongly enhanced by Hh treatment (Fig 6A, lane 2). This increased ubiquitination was abolished by down-regulation of Ubr3 (Fig 6A, lane 4), suggesting that Ubr3 mediates Hh induced ubiquitination of Cos2. This implied that Ubr3-mediated ubiquitination of Cos2 was tightly controlled by Hh signaling. Because Ci is not expressed in S2 cells, Hh-induced ubiquitination of Cos2 cannot be mediated by a positive, transcriptional feedback loop that depends on Ci. We therefore tested whether Hh may promote binding of Ubr3 to Cos2. We performed co-IP assays between the Ubr3 and Cos2 in the presence or absence of Hh. As shown in Fig 6B, the interactions between Ubr3 full-length protein and Cos2 (lanes 1 and 2 in Fig 6B) were strongly increased by Hh. These data show that Hh induces the ubiquitination of Cos2 by promoting the association of Ubr3 with Cos2. Consistent with Hh-induced poly-ubiquitination of Cos2, we also observed a faster degradation of Cos2 upon Hh treatment (Fig 6C and 6C').
the absence of signal in GFP control samples (lane 1) or upon omitting E1 and E2 enzymes. The membranes were stripped and re-probed with anti-GFP antibodies to ensure the presence of the UBR::GFP or GFP. An additional band of~80kDa is observed in UBR::GFP samples, which is approximately twice the size of UBR::GFP (arrow). (C) UBR domain interacts with endogenous Ubr3. UBR::GFP or GFP alone was immunoprecipitated from lysates of transiently transfected S2 cells using anti-GFP beads. Western blot was then performed with anti-Ubr3 antibody. Over-expressed UBR::GFP (lane 4), but not GFP alone (lane 3), co-immunoprecipitates with endogenous Ubr3 full length protein. doi:10.1371/journal.pgen.1006054.g004 Ubr3 Regulates Hh Signaling through Ubiquitination of Costal-2 and Kif7

Ubr3 positively regulates Shh signaling in zebrafish
To determine whether Ubr3 plays a conserved function in vertebrates, we created two independent zebrafish ubr3 mutant alleles using CRISPR/Cas9. The ubr3 gene is predicted to encode a protein of 1808 amino acids, and the ubr3 b1250 allele lacks 28 nucleotides ( Using an anti-Ubr3 antibody, we detected expression of Ubr3 in the developing retina, central nervous system and trunk, which are lost in ubr3 b1250/ b1251 mutant zebrafish (Fig 7A and 7B). Three independent crosses between single carriers heterozygous for the b1250 and b1251 alleles resulted in progeny with a distinguishable and reproducible retinal phenotype in a Mendelian frequency (f = 0.22, f = 0.27, f = 0.23, n = 270). At the 5-6-somites stage, phenotypically wild-type siblings display optic vesicles characterized by a compacted and stratified epithelium (Fig 7C, 7E and 7G). The optic vesicles of the ubr3 transheterozygous mutants failed to form a cohesive and stratified epithelium (Fig 7D, 7F and 7H). Because appropriate levels of Sonic Hedgehog (Shh) signaling are essential for eye morphogenesis [60,61], we examined the transcriptional levels of ptch2. In zebrafish, ptch2 is a direct target of Shh signaling [62,63]. In wild-type embryos, a gradient of ptch2 expression was observed within the optic vesicle (dotted area in Fig 7G and 7I). This gradient was characterized by high levels of ptch2-expressing cells localized in the ventral border of the vesicle, and low level expressing cells localized in the dorsal border region and vesicle core (Fig 7I). In ubr3 mutants, ptch2 expression was strongly decreased (Fig 7H and 7J). Consistent with decreased Shh signaling, ubr3 b1250/b1251 trans-heterozygous mutants show a 30% increase in the angle of agarose followed by western blot assays on the cell lysates. Both UBR domain fragments (lane 2) and the Ubr3 full length protein (lane 3) co-precipitate with Cos2, whereas GFP (lane 1) does not. S2 cells cotransfected with HA::Cos2 and Ubr3::GFP were treated with Colchicine for 5 hours prior to harvest. Coimmunoprecipitation assay with anti-GFP agarose beads and western blots were performed from the cell lysate. (D) Schematic diagram of Cos2 deletion constructs. (E) Co-immunoprecipitation assays with lysates from S2 cells, transfected with UBR::GFP and the constructs indicated in C reveal that the UBR domain of Ubr3 interacts physically with full length Cos2 and Cos2ΔC1-ΔC3 (red bars in C, lanes 1, 4-6 in D), but not   restricts CNS dorsal fate acquisition [69], this result supports the interpretation that Hedgehog signaling is decreased in ubr3 mutants. This finding is also consistent with our observation of decreased retinal ptch2 expression in the absence of ubr3 (Fig 7H and 7J). Because Kif7depleted zebrafish embryos do not show de-repression of Hh target genes in the CNS [27], our findings further suggest that, at least in zebrafish, Ubr3 may regulate not only Kif7 but also other intracellular negative regulators of Hedgehog signaling in the CNS. Different zebrafish Hh signaling mutants show distinct degrees of severity, highlighting the tissue-specific requirements of Hh levels during development [60,[70][71][72][73][74]. Similarly, loss of ubr3 does not result in cyclopia or inner ear defects, showing that these mutants have a less severe phenotype when compared to smoothened mutant animals. Hence, ubr3 zebrafish mutants retain some residual Hh signaling. Thus, our data show that Ubr3 positively regulates Hedgehog signaling in tissues sensitive to high levels of Hh like the mesoderm and neuroectoderm. In addition, the transcription of ubr3 is strongly reduced in smo hi1640-/mutant animals [70], which lose Shh activity (S5F and S5H Fig

UBR3 negatively regulates protein level of Kif7 through polyubiquitination in mammalian cells
To test whether UBR3 also plays a role in Shh signaling in mammals, we used C3H10T1/2 mouse mesenchymal cells. These cells respond to Shh and activate Shh target genes [76]. We first confirmed that Ubr3 is expressed in C3H10T1/2 cells by RT-PCR (see Fig 8B'). We then infected these cells with a lentivirus bearing 7 tandem binding sites for Gli (the vertebrate homologue of Ci) that control the expression of a GFP reporter. Addition of either the Shh ligand or purmorphamine, an agonist of Smo [77], to C3H10T1/2 cells induced GFP expression in about 25% of the cells (Fig 8A and 8A'). To determine whether knockdown of UBR3 impairs Shh signaling, we measured the proportion of GFP-expressing C3H10T1/2 cells transfected with one of four different siRNAs against UBR3 or a scrambled siRNA control, followed by purmorphamine treatment. Induction of the Gli::GFP reporter by purmorphamine was suppressed when siRNA reduced the UBR3 levels (Fig 8B), as judged by real time PCR (Fig 8B'). In addition, down-regulation of UBR3 resulted in up-regulation of Kif7 (Fig 8C), the mammalian homolog of Cos2. To assess poly-ubiquitination of Kif7, we purified Kif7 through immunoprecipitation and loaded the Western blot lanes with equal amounts of protein (unlike in Fig 8C where we loaded equal amounts of cells). We observed decreased poly-ubiquitination of Kif7 upon knockdown of UBR3 (Fig 8D). These data indicate that UBR3 regulates Shh signaling through polyubiquitination of Kif7 in vertebrate cells, a process that seems to be evolutionarily conserved.

Discussion
Numerous studies have shown that Cos2 plays a central role in Hh signaling [10,11,22,25,26,78,79]. Cos2 is both necessary and sufficient to regulate Ci [11,22] and the level of Cos2 protein is critical for activating Hh signaling [80,81]. Here, we identified Ubr3 as a novel regulator of Cos2 in a forward genetic screen in Drosophila and showed that this gene is conserved in vertebrates and affects Hh signaling. We present evidence that the level of Cos2 protein is tightly controlled through a Ubr3-mediated poly-ubiquitination pathway (Fig 8E).
Although most of the core components of Hh signaling are evolutionarily conserved, there are differences in Hh signaling between vertebrates and invertebrates [82]. For example, Cos2 can be phosphorylated by the kinase Fused [30,31], but the kinase that phosphorylates Kif7 remains to be identified, because mice lacking Fused have no apparent defects in Hh signaling [83,84]. Given that a Kif7 phosphatase affects Hh signaling in vertebrates [85] it is likely that phosphorylation of Kif7 is important even if these sites are different than those observed in Cos2. Here, we present the first evidence that the levels of Cos2 and Kif7 proteins are also controlled by poly-ubiquitination via a conserved Ubr3 E3 ligase. The conservation of this mechanism is supported by the finding that fly Cos2 rescues the Kif7 mutant phenotypes in zebrafish [27]. Although we have shown that the degradation of Cos2 protein is regulated by Ubr3 mediated ubiquitination, the increased Cos2 proteins in ubr3 mutant cells may also result from upregulated transcription of Cos2.
Although Hh promotes poly-ubiquitination and degradation of Cos2 (Fig 6A and 6C), we did not observe a decrease of Cos2 proteins at the morphogenetic furrow, where Hh signaling is activated. Instead, the level of the Cos2 protein is modestly elevated when compared to surrounding tissues/cells (S6A and S6A' Fig), consistent with a previous finding [11]. We also observe that activation of Hh in S2 cells up-regulates Cos2 (Fig 6C). The observation that activation of Hh signaling promotes the degradation of Cos2 and that Cos2 protein level is increased, but not decreased by Hh activation, suggests that some mechanism other than ubiquitination upregulates the level of Cos2 protein (Fig 8F). Ubr3-mediated degradation of Cos2 may function as a mechanism to prevent aberrantly high levels of Cos2, thereby toning down Hh signaling. This may also underlie the observation that not all cells that require Hh signaling are affected in flies. This hypothesis is also supported by the finding that loss of ubr3 in zebrafish affects developmental processes that rely on high levels of Shh signaling but does not affect those that respond to low Shh signaling (Fig 7 and S4 Fig).
Cul1 functions downstream of Cos2 to process Ci 155 , one would anticipate that Cul1 is epistatic to Cos2. This is inconsistent with the observation that down-regulation of Cul1 in ubr3 clones in the morphogenetic furrow of Drosophila eye discs fails to restore Ci 155 expression (Fig 3E), whereas down-regulation of Cos2 restores Ci 155 levels ( Fig 3D). This may be because the RNAi expression does not deplete the protein sufficiently, or because, Cos2 may regulate Ci 155 through a mechanism independent of Cul1.
Although our data clearly show that Ubr3 plays a role in Hh signaling at the morphogenetic furrow, we do not observe a loss of Ci 155 in ubr3 mutant clones in wing discs (S6B and S6B ' Fig). However, we observed a similar up-regulation of Cos2 in ubr3 mutant clones in wing discs (S6C and S6C' Fig), implying that Ubr3 mediated poly-ubiquitination of Cos2 may be present in wing discs. The lack of a Hh phenotype in posterior compartment cells of wing discs may be due to another E3 ligase that is functionally redundant and downregulates Cos2. Alternatively residual Ubr3 in ubr3 mutant cells due to perdurance of Ubr3 products may partially downregulate Cos2, allowing activation of Hh signaling. When we sensitized the background by over-expressing ptc DN to ectopically activate Hh signaling, we find that loss of ubr3 strongly suppresses the activation of Hh signaling in clones, gauged by the reduced clone sizes and Ci 155 levels (S6D-S6E ' Fig). This may also be the reason why not all tissues display the typical Shh phenotype in zebrafish. In addition, ectopic activation of Hh signaling leads to up-regulated transcription of ubr3 (S6F-S6J Fig), suggesting that the positive feedback of Ubr3 is present in the wing.
Hh signaling shares many similarities with Wnt signaling [86]. Both pathways regulate many developmental processes and induce human cancers when the pathways are aberrantly activated. Moreover, the principal signaling mechanisms are based on similar features. Each pathway is activated through ligand binding of a G-protein coupled receptor, leading to the downstream activation of a transcription factor through phosphorylation-dependent proteolysis. Axin is the scaffold protein that recruits an activation complex in Wnt signaling, which mediates phosphorylation of β-catenin [87]. This function is similar to that of Cos2 in Hh signaling. Interestingly, previous studies have shown that the levels of Axin protein are also regulated by an E3 ligase, RNF146, through poly-ubiquitination [88][89][90]. Upon activation of Wnt signaling, Axin undergoes tankyrase-dependent poly ADP-ribosylation, which promotes RNF146-Axin interaction [89]. Ubr3 seems to regulate the poly-ubiquitination of Cos2 in a similar manner, given that Hh activation promotes the Ubr3-Cos2 interaction and the ubiquitination of Cos2. Hence, our data suggest further similarities between the Hh and Wnt signaling pathways.

Zebrafish strains and husbandry
Zebrafish strains were AB wild-type, ubr3 b1250 , ubr3 b1251 and smo hi1640 . The ubr3 mutations are recessive alleles. Phenotypically wild-type siblings were used as controls and labeled as wild type in Fig 7. Animals were raised in a 10 hour dark and 14 hour light cycle and maintained as previously described [99]. Embryos were staged according to the standard series [100]. All animal use protocols were IACUC-approved.

CRISPR mutagenesis and genotyping
CRISPR mutagenesis was carried as previously described with minor modifications [101,102]. The zebrafish ubr3 reference sequence used in this study was XM_009304449.1. Identification of target sequences was done using Zifit software [103,104]. Candidate sequences were then blasted against the zebrafish genome (Zv9) and those with unique hits were selected. The following target sequences were selected b1250: 5'-GGGGCCTGTGACTGCGGGGA-3', located in the sense strand, and b1251: 5'-GGCGTTATCGTAGGATCGGA3', located in the antisense strand (Fig 7, S1 Fig). A guide RNA (gRNA) template was created by PCR. A T7 promoter site was incorporated in the gene specific oligonucleotide, followed by the target sequence and the start of the guide RNA sequence (5'-gttttagagctagaaatagc-3'). The complementary guide RNA scaffold oligonucleotide sequence used was 5'-gatccgcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac-3'. PCR was performed using Phusion polymerase (NEB) following the manufacturer's recommendations. 10μM of each primer was used for the reaction. The first denaturation step was carried out at 98°C for 30 sec, followed by 40 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 15 seconds. A final extension step was introduced at 72°C for 10 minutes. PCR products were purified using a PCR purification kit (QIAGEN). RNA was transcribed using a MEGAscript T7 kit following the manufacturer recommendations. A volume of 2nl of Cas9 RNA and gRNA were co-injected at a concentration of 100ng/μl each.
Screening of F0 founders and genotyping of F1 carriers were done by PCR and sequencing using the following primers: Embryo manipulation and RNA injection 250 pg of RNA encoding for a dominant negative regulatory subunit of the Protein Kinase A (dnPKA) [75] was injected into 1 cell stage embryos. dnPKA construct was linearized with NotI and transcribed with SP6 using a mMessage mMachine kit following the manufacturer´s recommendations. Embryos were fixed at 27hpf and processed for in situ hybridization against ubr3.
Zebrafish immunolabeling was performed as previously described [111] with the following minor modifications. 18-somites stage embryos were fixed in BT-fix overnight at room temperature. Embryos were permeabilized in PBS+1% Tween20 for 5 hours at room temperature. Anti-UBR3 antibody (Sigma Prestige, catalogue #HPA035390) was diluted in 1/500. Biotinylated anti-rabbit was used at 1/500. To detect signal, ABC kit (Vectorlabs) was used. A and B reagents were mixed together at an 1/100 dilution in PBS-Block and pre-incubated for 20 minutes at room temperature, then added to the samples for 25 minutes. Tyramide from the TSA kit (Perkin-Elmer) was diluted 1:50 in pre-warmed buffer reagent, and added to samples for 20 minutes following the manufacturer's recommendations. The detection reaction was stopped by adding cold PBS+0.1%Tween20, followed by 4 washes in PBS+0.1%Tween20.

Microscope image acquisition
Images were acquired using LSM510 and LSM710 confocal microscopes (Zeiss) and examined and processed using LSM viewer (Zeiss), ZEN (Zeiss) and Photoshop (Adobe) software. Immunostained zebrafish embryos were immersed in Vectashield, mounted laterally in a slide chamber and imaged with a Zeiss LSM5 confocal microscope. Live embryos were mounted laterally in 3% methylcellulsose and imaged on a compound microscope using DIC. ISH treated embryos were dissected in 90% glycerol, flat-mounted in 100% glycerol in a slide chamber and imaged on a compound microscope using DIC.

Cloning, plasmid constructs and antibody production
A ubr3 genomic rescue construct was constructed by cloning a 18.3 kb fragment of genomic DNA that contains the ubr3 gene (X: 7,935,666. . . 7,953,967) [Release 6 Drosophila reference genome, [112]] into P[acman] [92,113]. ubr3 cDNA was constructed from exon sequences and cloned into pUASTattB using a GENEART Seamless Cloning and Assembly Kit (Life Technologies). GFP was tagged to the carboxyl terminus of the full length ubr3 sequence or a partial sequence encoding only the UBR domain (aa 222-292). The flag sequence was conjugated to the carboxyl terminus of the full length ubr3 sequence in the primer. Ubr3::flag was then amplified through PCR and cloned into pUASTattB through XhoI and XbaI. To generate E3 dead form of flag tagged Ubr3 expression construct, mutations results in all residues shown in red box in Fig 4A changed to alanines were introduced through synthesized DNA which spans 500 bp downstream from RsrII. This synthesized DNA fragment was then cloned together with PCR amplified flag tagged carboxyl fragment of Ubr3 into pUASTattB-Ubr3-flag through RsrII, BbsI and XbaI. The HA::Cos2N1 to 3 constructs were cloned into pUASTattB through EcoRI and XbaI. HA::Cos2ΔN1-2, HA::Cos2ΔC1-3 have been described [114]. Myc:: Cos2 was constructed by fusion of 5xMyc tags to the N-terminus of the Cos2 coding sequence. The HA::Ub transgene has been described previously [34]. Kif7::GFP construct is a gift from Dr. Chi-Chung Hui [78].
The 7Gli:GFP reporter of Hedgehog signaling activity contains 7 repeats of the Gli binding site (5'-TCGACAAGCAGGGAACACCCAAGTAGAAGCTC) followed by GFP [115]. The primer pair (forward 5'-TGAAGCTTGCATGCCCTGCAGGACAAGCAGGGAACGCCCA AGTAG and reverse 5' CTCGAGTACCGGATCCATTATATACCCTCTGCAGACTTGG GTGTTCCCTGCTTGTCG) was used to amplify the Gli binding sequences from the 8Gli-Luc plasmid by PCR. The reverse primer also contains a TATA sequence, which was used to rebuild the TATA box after the Gli binding sites. The destination plasmid pRRL.sin-18.ppt.TCF/LEF: GFP.pre [116] was linearized by PstI and BamHI digestion to cut out the TCF/LEF sequence and the TATA box. The resulting products were recombined into a linearized destination plasmid by infusion cloning according to the manufacturer's protocol (Clontech).
For Ubr3 antibody production, the sequence encoding aa 751-1500 of Ubr3 was cloned into pET21 expression construct and expressed in E. coli. Purified inclusion bodies were used to immunize guinea pigs.
In situ hybridization ubr3 in situ hybridization probes I and II anti-sense sequences contain 2558 to 3576 nt and 3775 to 4770 nt of ubr3 cDNA, respectively. Anti-sense sequences were cloned into pGEM-T vector (Promega). Before transcription, the construct was linearized by SalI. ubr3 RNA in situ probes were transcribed and labeled with a digoxigenin [113] RNA labeling kit (Roche). In situ hybridization to whole-mount discs was performed as previously described [117]. Probe I was used in images shown in Fig 2F,  Zebrafish whole-mount in situ hybridization was carried out as described with minor modifications [118]. Digoxigenin-labeled probes were prepared according to manufacturer´s instructions (Roche). Probe signal was detected using NBT/BCIP mix (Roche). The ptch2 probe was kindly shared by Stone Elworthy (University of Sheffield, UK).
Cell culture assays S2 cells were cultured at 25°C in Schneider's medium (Life Technologies) plus 10% heat-inactivated fetal bovine serum (Sigma), 100 U/mL penicillin (Life Technologies), and 100 μg/mL streptomycin (Life Technologies). Cells were split every 3 days and plated at a density of 10 6 cells/well in 12-well cell culture plates for experiments. Transfections were carried out using Effectene transfection reagent (Qiagen). Ubr3 dsRNA was synthesized against nt 652-1,191. dsRNAs transfection and dsRNA against GFP have been described [114]. CHX (100 μM, Sigma) and MG132 (50 μM, Sigma) in dimethyl sulfoxide (DMSO) were added to S2 cells 48 hours after transfection and incubated for indicated time. An equal amount of DMSO was added as a negative control (-). 1ug/ml Colchicine (sigma) was incubated with cells for 5 hours before harvest. C3H10T1/2 cells were cultured at 37°C in 5% CO 2 in air in Eagle's Basal medium with Earle's BSS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10% fetal bovine serum, as described by the ATCC (http://www.atcc.org/). Cells were split when reaching 80-90% confluence. Cells were transduced with lentivirus containing Gli::GFP reporter construct for 16h and then plated on 12-well cell culture plates. On the second day, siRNAs were incubated with Lipofectamine RNAiMAX reagent (Invitrogen) overnight. The sequences of siRNAs against ubr3 are as follows: siRNA 1: GTTATAGCTTTGAATCAGT; siRNA 2: CAGAGTTTGCCT CACGACA; siRNA 3: CAAGATTGGTTTGATGCTA; siRNA 4: CAGAAATTGCTCGCA GAGT. Stealth RNAi™ siRNA Negative Control Med GC Duplex (Invitrogen) was used as control siRNA. 3μg/ml Shh (R&D systems) or 10 μM purmorphamine (Calbiochem) in culture medium, or the same amount of vehicle in culture medium for uninduced controls, was added to cells on the third day. Cells were photographed for GFP fluorescence and harvested 48 h after purmorphamine induction. The number of GFP-positive cells was manually counted and statistical testing was performed with a one way ANOVA followed by Dunnett's test using uninduced cells as a control.

Quantitative real-time PCR
For RNA extraction, total RNA from C3H10T1/2 cells was isolated by using Absolutely RNA miniprep Kit (Agilent Technologies). cDNA was synthesized using Superscript III First Strand Synthesis System for RT-PCR (Invitrogen). Quantitative real-time PCR (qPCR) was conducted with a Master SYBR Green kit (Applied Biosystems) and gene-specific primer sets on a Step One Plus real-time PCR system (Applied Biosystems). Each experiment was performed with three biological sample repeats and each PCR was performed in triplicate. L19 was used as an endogenous reference. The gene-specific primer sets used were as follows: L19 (RpL19): 5'-GGTCTGGTTGGATCCCAATG-3' and 5'-CCCGGGAATGGACAGTCA-3'; UBR3: 5'-CTGATTCATAGAGGAGGCAG-3' and 5'-ATGGAACAGCTGATTCAGAC-3'.

In vitro ubiquitination assay
In vitro auto-ubiquitination assays were performed as described previously [119] with modifications. In brief, S2 cells were first transiently transfected with GFP, UBR-GFP or Ubr3-GFP. S2 cell cultures were collected 48 hours post-transfection and lysed on ice for 45 minutes under stringent conditions to minimize interactions with other proteins, using 100 μl RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing 1x Complete protease inhibitors cocktail (Roche) for every 10 6 cells seeded. The lysates were then added to 30 μl bed volume of Chromotek GFP Trap beads (Allele Biotechnology), previously equilibrated with RIPA buffer, and incubated by rocking at 4°C for 3 hours. After washing with RIPA buffer, 20% of the beads were retained for assessing the expression of GFP protein by Western blot analysis using anti-GFP (1:1000, Zymed). The remainder of the lysates were equilibrated by rinsing twice in 1x Ubiquitination Reaction buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 50 mM NaCl, 1 mM dithiothreitol DTT, 1x protease inhibitors cocktail). The ubiquitination reaction was assembled by adding rabbit UBE1 E1 (Boston Biochem Cat. #302) and human recombinant His 6 -hHR6A E2 (Boston Biochem Cat. E2-612) conjugating enzymes and FLAG-Ubiquitin (Sigma) on ice and incubated at 30°C for 30 minutes. The reactions were stopped by adding 1x Laemmli buffer, after which the samples were boiled for 10 minutes and analyzed by SDS-PAGE and Western blot using anti-FLAG M2 monoclonal antibody (1:1000, Sigma).
For Cos2 ubiquitination assays, S2 cell culture and RNAi were performed as described previously [114]. Transfections were carried out using Effectene transfection reagent (Qiagen). The immunoprecipitation and immunoblot analysis were performed using standard protocols. Myc-Cos2 was constructed by fusion of 5xMyc tag to the N-terminus of Cos2 coding sequence. HA-Ub and Ub mutants have been described [34]. The HA::UbK48 only has mutations at all of the lysine residues with the exception of K48. GFP RNAi has been described. Ubr3 dsRNA was synthesized against nucleotides 652-1191. The following antibodies were used: mouse anti-Myc (1:5000, 9E10, Santa Cruz), anti-GFP (1:1000, Millipore), anti-HA (1:5000, F7, Santa Cruz), and anti-β-tubulin (1:2000, E7, DSHB). . Arrow in A' shows moderate increase of Cos2 protein levels in the morphogenetic furrow. (B-B') Wing disc from 3 rd larvae with ubr3 B mutant clones (green) was stained with anti-Ci 155 (red) and anti-senseless (sens, cyan). (C-C') Wing disc from 3 rd larvae with ubr3 B mutant clones (green) was stained with anti-Cos2 (red) and anti-senseless (sens, cyan). (D-D') A wing disc from 3 rd larvae with ptc DN expressing clones (green) was stained with anti-Ci 155 (red) and anti-senseless (sens, cyan). (E-E') Wing disc from 3 rd larvae with ubr3 B mutant clones expressing ptc DN (green) was stained with anti-Ci 155 (red) and anti-senseless (sens, cyan). (F, H-J) In situ hybridizations were performed on wing discs from 3 rd instar larvae with indicated genotypes using an anti-ubr3 probe. (G) A wing disc from 3 rd larvae in which eyg-Gal4 driven expression of GFP was labeled with anti-GFP (green in G, indicating eyg-Gal4 expression region), anti-Sens (cyan in G, labeling wing margin) and anti-Ci 155 (red in G). Arrows indicate elevated ubr3 transcription in eyg-Gal4 expressing domain. (TIF)