EDD, a Ubiquitin-protein Ligase of the N-end Rule Pathway, Associates with Spindle Assembly Checkpoint Components and Regulates the Mitotic Response to Nocodazole

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G(2)/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis

Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue, Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals

Regolazione ubiquitina-proteasoma dipendente della stabilità proteica di p73

Background and aim p73 is a structural and functional homologue of the tumor suppressor transcription factor p53, which binds to canonical p53 DNA-binding sites, activates transcription from p53-responsive promoters and, hence, induces cell cycle arrest and apoptosis (reviewed in Melino et al., 2002). In contrast to p53, p73 exists as several distinct protein isoforms (-) generated by alternative splicing at the C-terminal (De Laurenzi et al, 1998, 1999 and 2000). Additionally, the p73 gene has two distinct promoters; the first promoter (P1) yields proteins possessing an N-terminal transactivation domain (TAD), the transcriptionally active (TA) isoforms. The usage of the alternative internal promoter (P2) gives rise to N-terminally truncated proteins (N isoforms), which lack the TAD and, as a result, act as dominant negative inhibitors of p53 and TAp73 tumor suppressive functions (Kaghad et al., 1997; Ueda et al., 1999). Thus, TA- and Np73 isoforms display antagonistic functions: the TAp73 variants largely mimic p53 suppressive activities, while the Np73 proteins promote cell survival and exhibit oncogenic properties (Yang et al., 2000a; Grob et al., 2001; Sayan et al., 2004). As a result of the opposite activities exerted by TA- and Np73 proteins, the balance between cell death and survival, particularly in cells harboring p53 mutations, will crucially depend on the relative proportions of the two isoforms (Melino et al., 2002). Similarly to p53, p73 expression is maintained at low levels in mammalian cells, and its cellular induction and activation is mainly controlled at the post-translational level. p73 is polyubiquitylated in vivo and degraded via the proteasomal proteolytic system (Bernassola et al., 2004; Maisse et al., 2004). It has been previously reported that p73 ubiquitylation is catalysed by the HECT type E3 ubiquitin ligase (E3) Itch (Rossi et al., 2005). In unstressed cells, Itch targets both TA- and Np73 for protein ubiquitylation, thereby keeping their expression levels low under normal conditions. Following DNA damage treatment, TAp73 protein levels accumulate, while Np73 is rapidly degraded, in an Itch-independent manner. In several tumor cell lines, the induction of TAp73 in response to chemotherapeutic drugs is, at least partially, accomplished through Itch downregulation (Rossi et al., 2005). Our findings imply that different E3 ligases can account for p73 degradation in different conditions. On the basis of these evidences, my PhD project has been focused on ubiquitin-dependent degradation of p73, by one side testing the possible implication of another E3 ligase activity in the regulation of p73 protein level, and by the other analyzing the molecular mechanisms of Itch self- ubiquitylation and investigating its possible involvement in the regulation of Itch protein stability. Results Self-ubiquitylation activity of E3 ligases (E3s) has been previously described for both RING-type and HECT-type E3s (Bruce et al., 2008). It is thought to mainly act as a regulatory mechanism that controls the abundance of E3s by marking them for degradation (Yang et al, 2000b; Fang et al, 2000). It has been previously reported that Itch is capable to undergo self-ubiquitylation although its physiological role has not been clearly elucidated (Gao et al., 2004; Mouchantaf et al., 2006). In the context of the first part of my project, we tested whether Itch self-ubiquitination could affect its protein stability. We demonstrated that Itch generates self-assembled Lysine-63 linked polyubiquitin chains, a signal generally not involved in targeting proteins for proteasome-dependent degradation. Consistently with this, we shown that Itch is a high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrated that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is indistinguishable from the one of the wild-type protein. All these results demonstrate that Itch self-ubiquitylation activity does not regulate its protein stability. As discussed above, the evidence that Itch is responsible for keeping both TAp73 and Np73 levels low under normal condition but not in response to DNA damage suggests the involvement of another pathway to target p73 for degradation (Rossi et al., 2005). Additionally, it has been described that in C. elegans, the regulation of the p53-like protein CEP-1 is controlled by the F-box protein named FSN-1 (Gao et al., 2008). Among 520 genome-predicted F- box proteins, FSN1 is one of the few to be conserved through evolution, and its human ortholog is FBXO45. F-box proteins represent the substrate targeting subunit of a class of RING-type E3 ubiquitin ligases known as Skp1-Cul1-F-box complexes (SCF). Taken together, these data led us to test the involvement of SCFFBXO45 complex in the regulation of p73 stability. We firstly proved the existence of a functional relation between SCF complex and p73 by demonstrating that the expression of the dominant negative of Cul1, but not the other cullins, stabilizes p73. Subsequentially, we found that SCFFBXO45 specifically interacts with p73, both TAp73 and Np73 isoforms, suggesting that, similarly to Itch, FBXO45 is not able to discriminate between these two. Given that the major function of F-box proteins involves the ubiquitination of their target proteins, we sought to determine whether FBXO45 ubiquitinates p73 in mammals cells. We demonstrated indeed that FBXO45 significantly stimulates the ubiquitination of p73, both in vitro and in vivo. Significantly, on one hand the overexpression of FBXO5 induces the proteasome-dependent degradation of p73, and on the other the silencing of FBXO45 through RNA interference results in accumulation of p73. Moreover we found that FBXO45, similarly to Itch, is down-regulated in response to DNA damage, allowing thus p73 levels to increase in response to stress. Conclusions p73, a member of the p53 family, is a transcription factor controlling different biological processes, including cell death, tumorigenesis and neuronal differentiation. Knowledge on the mechanisms regulating p73 levels in basal conditions as well as in response to stress is essential to design new therapies that require p73 induction. Our group has previously demonstrated that the HECT E3 ubiquitin ligase Itch is capable to polyubiquitylate p73 and induce its degradation in a proteasome-dependent manner. The work performed in this PhD project has been focused on ubiquitin-dependent degradation of p73, analyzing the molecular mechanisms of Itch self- ubiquitylation and its possible role in Itch protein stability, and testing the possible implication of an E3 ubiquitin ligase, different from Itch, in the regulation of p73 protein level. In this study, we provide evidences that Itch engages an intermolecular reaction generating Lys63 polyubiquitin chains, and that this auto-modification does not regulate Itch protein stability. Furthermore, our findings demonstrate that the self-polyubiquitin chains generated by Itch do not serve as either proteasome or lysosome targeting. Both mono- and poly-ubiquitylation of protein substrates have been associated with internalization, sorting and changes in their subcellular localization. Hence, ubiquitin conjugation might represent a signal for Itch to translocate to distinct cellular compartments, which ultimately, would modify its accessibility to certain substrate molecules. Itch is predominantly localized to early and late endosomal compartments and lysosomes. Numerous Itch substrates are transcription factors mainly residing in the nuclear compartment. Hence, self-ubiquitylation may represent an auto-regulatory mechanism controlling Itch cytoplasmic-nuclear shuffling. The F-box proteins recruit specific substrates to the E3 ligase SCF complex, thus targeting them to proteasome-dependent degradation. Despite the large number of F-box proteins, only nine human SCF ubiquitin ligases have well-established substrates. Here we show that the F-box protein FBXO45 is recruited into SCFFbx45 complex and is able to bind to p73 promoting its ubiquitylation and its proteasome- dependent degradation. Since FBXO45 depletion sensitizes cell to apoptosis, we elucidate a new, conserved mechanism that could be potentially used to develop new strategies aimed to potentiate the apoptotic response of cancer cells following chemotherapy. p73 è un omologo strutturale e funzionale del soppressore tumorale p53, in grado di legare siti canonici di binding riconosciuti da p53, attivare la trascrizione attraverso promotori responsivi a p53, e quindi indurre arresto del ciclo cellulare e apoptosi (funzioni riassunte in Melino et al., 2002). Al contrario di p53, p73 possiede diverse isoforme (-) generate attraverso splicing alternativo all'espremità C-terminale (De Laurenzi et al, 1998, 1999 and 2000). Inoltre, il gene TP73 presenta due distinti promotori, il primo dei quail (P1) genera delle isoforme che contengono all'N-terminale il dominio di transattivazione (TAD), e che sono dunque in grado di indurre transattivazione (isoforme TA). L'utilizzo di un promotore alternativo interno (P2) produce isoforme tronche all'estremità N- terminale (isoforme N), che sono prive del TAD e dunque agiscono come inibitori dominanti negativi delle funzioni di soppressori tumorali ascrivibili a p53 e a p73 (Kaghad et al., 1997; Ueda et al., 1999). Dunque, le isoforme TAp73 e Np73 mostrano funzioni opposte: le isoforme TAp73 mimano le attività di p53, mentre le isoforme N promuovono la sopravvivenza cellulare e mostrano proprietà oncogeniche (Yang et al., 2000a; Grob et al., 2001; Sayan et al., 2004). Ne risulta che l'equilibrio tra sopravvivenza e morte cellulare, in particolare in cellule che possiedono mutazioni per p53, dipende dal rapporto tra le due isoforme (Melino et al., 2002). Come p53, l'espressione di p73 è manenuta a livelli bassi nelle cellule di mammifero, e la sua induzione e attivazione è prevalentemente controllata a livello post-traduzionale. p73 è poliubiquitinata in vivo e degradata attraverso il proteasoma (Bernassola et al., 2004; Maisse et al., 2004). È stato riportato che l'ubiquitinazione di p73 è catalizzata da Itch, una E3 ubiquitina ligasi (E3) di tipo HECT (Rossi et al., 2005). In condizioni normali. Itch ubiquitina sia TAp73 che Np73, mantenendo dunque bassa la loro abbondanza nelle cellule. A seguito di stress genotossico, i livelli proteici di TAp73 aumentano, mentre Np73 è rapidamente degradato, in maniera Itch- indipendente. In diverse linee tumorali, l'induzione di TAp73 in risposta a chemioterapici è ottenuta almeno parzialmente attraverso la downregolazione di Itch (Rossi et al., 2005). I nostri precedenti risultati suggeriscono dunque che diverse E3 ligasi possano essere responsabili della degradazione ubiquitina-dipendente di p73 in differenti condizioni. Sulla base di ciò, il mio progetto di dottorato ha riguardato la degradazione ubiquitina-dipendente di p73, da una parte testando l'ipotesi dell'esistenza di di un'altra E3 ligasi la cui attività sia implicata nella regolazione dei livelli di p73, e dall'altra analizzando i meccanismi molecolari alla base dell'autoubiquitinazione di Itch e il suo possibile coinvolgimento nella regolazione della sua stabilità proteica. Risultati L'autoubiquitinazione di E3 ligasi è stata precedentemente descritta sia per ligasi di tipo RING che di tipo HECT (Bruce et al., 2008). Si ritiene che agisca prevalentemente come un meccanismo regolatorio che controlla l'abbondanza delle E3 marcandole per la degradazione attraverso il proteasoma (Yang et al, 2000b; Fang et al, 2000). È stato descritto che Itch ha la capacità di catalizzare la sua autoubiquitinazione, sebbene il suo ruolo fisiologico non sia stato chiarito (Gao et al., 2004; Mouchantaf et al., 2006). Nella prima parte del mio progetto di dottorato, ci siamo chiesti se l'autoubiquitinazione di Itch potesse influire sulla sua stabilità proteica. Abbiamo dimostrato che Itch è in grado di generare su un'altra molecola di Itch catene di poliubiquitina costruite attraverso la Lisina 63 presente nell'ubiquitina. Catene di poliubiquitina così assemblate costituiscono un segnale che non è coinvolto nella marcatura delle proteine per la degradazione proteasoma-dipendente. Coerentemente con ciò, abbiamo dimostrato che Itch è una proteina molto stabile, i cui livelli sono sono modificati in maniera significativa da trattamento con inibitori del proteasoma o del lisosoma. Inoltre, abbiamo dimostrato che il tasso di decadimento del mutante di Itch cataliticamente inattivo (ovvero incapace di autoubiquitinazione), è indistinguibile da quello della proteina wild type. Tutti questi risultati dimostrano che l'autoubiquitinazione di Itch non regola la stabilità proteica di questa E3 ubiquitina ligasi. Come già discusso, il fatto che in condizioni normali Itch sia responsabile del mantenimento a bassi livelli sia di TAp73 che di Np73, ma non in seguito a danno al DNA, suggerisce il coinvolgimento di almeno un altro pathway responsabile della degradazione di p73 (Rossi et al., 2005). Inoltre, è stato descritto che, in C. elegans, la regolazione dell'ortologo di p53, CEP-1, è controllata da una proteina F-box denominata FSN1 (Gao et al., 2008). Tra circa 520 F-box protein predette nel genoma di C. elegans, FSN-1 è una delle poche conservate attraverso l'evoluzione, e il suo ortologo umano è FBXO45. Le proteine F-box rappresentano la subunità responsabile del riconoscimento specifico del substrato di una sottofamiglia di E3 di tipo RING nota come complesso Skp1- Cul1-F-box (SCF). Tutti insieme questi dati ci hanno spinto a indagare il possibile coinvolgimento del complesso SCFFBXO45 nella regolazione della stabilità di p73. In primo luogo abbiamo verificato l'esistenza di una relazione funzionale tra il complesso SCF e p73 dimostrando che l'espressione del mutante dominante negativo di uno dei componendi dell'SCF, la cullina Cul1, stabilizza p73, al contrario delle altre culline. Successivamente, abbiamo dimostrato che SCFFBXO45 interagisce in maniera specifica sia con TAp73 che con Np73, suggerendo che, in maniera simile ad Itch, FBXO45 non è in grado di distinguere tra le due isoforme. Poiché la funzione principale delle proteine F-box riguarda l'ubiquitinazione dei loro substrati, abbiamo verificato se FBXO45 ubiquitinasse p73 nelle cellule di mammifero. Abbiamo dimostrato che FBX045 stimola significativamente l'ubiquitinazione di p73, sia in vitro che in vivo. Inoltre, abbiamo osservato che FBXO45, come Itch, è downregolata in risposta a danno al DNA, permettendo che i livelli di p73 aumentino. Conclusioni p73, un membro della famiglia di p53, è un fattore di trascrizione che controlla diversi processi biologici, tra cui la morte cellulare, la tumorigenesi e il differenziamento neuronale. Conoscere i meccanismi che regolano i livelli di p73 in condizioni basali e in risposta allo stress cellulare è essenziale per progettare nuove terapie che richiedano l'induzione di p73. Il nostro gruppo ha precedentemente dimostrato che l'E3 ubiquitina ligasi di tipo HECT denominata Itch è in grado di poliubiquitinare p73 ed indurre la sua degradazione attraverso il proteasoma. Il lavoro riguardante questo progetto di dottorato è stato incentrato da una parte sulla degradazione ubiquitina-dipendente di p73, analizzando i meccanismi molecolari dell'autoubiquitinazione di Itch e il suo possibile ruolo nella stabilità proteica di Itch stesso, e dall'altra testando il possibile coinvolgimento di una E3 ubiquitina ligasi diversa da Itch nella regolazione dei livelli proteici di p73. In questo lavoro, abbiamo dimostrato che Itch è impegnata in una reazione intermolecolare che genera catene di poliubiquitina sintetizzate impiegando la Lys63 dell'ubiquitina stessa, e che questa auto-modificazione non regola la stabilità proteica di Itch. Inoltre, i nostri dati dimostrano che l'auto-poliubiquitinazione di Itch non conduce Itch stessa alla degradazione proteasoma- o lisosoma- dipendente. Sia la mono che la poliubiquitinazione delle proteine è stata associata con internalizzazione, smistamento e con cambiamenti nella localizzazione subcellulare. Per cui l'ubiquitinazione di Itch potrebbe rappresentare un segnale per la sua traslocazione in altri compartimenti cellulari, e dunque modificare il suo accesso ad alcuni suoi substrati. Itch è infatti localizzata in maniera predominante negli endosomi precoci e tardivi e nei lisosomi. Numerosi substrati di Itch sono fattori di trascrizione che si trovano dunque prevalentemente nel compartimento nucleare. Quindi, l'autoubiquitinazione potrebbe rappresentare un meccanismo autoregolatorio che controlla il trasporto di Itch tra il citoplasma e il nucleo. Le proteine F-box reclutano substrati specifici al complesso E3 ligasi Skp1-Cul1-F-box (SCF) ed quindi alla degradazione proteasoma- dipendente. Malgrado il gran numero di proteine F-box esistenti, solo di nove di esse si conoscono approfonditamente i substrati. In questa tesi abbiamo dimostrato che la proteina F-box FBXO45, è parte di un complesso SCF ed è capace di legare p73, di cui promuove l'ubiquitilazione e la degradazione proteasoma- dipendente. Poiché il silenziamento di FBXO45 sensibilizza le cellule all'apoptosi, noi abbiamo descritto un nuovo meccanismo, conservato attraverso l'evoluzione, che può essere potenzialmente oggetto dello sviluppo di nuove strategie per aumentare la risposta apoptotica in cellule cancerose attraverso la chemioterapia

Sgg regulates <i>hh</i>-<i>lacZ</i> expression in both the posterior and anterior compartments.

<p>(A-U) Confocal images of EA disc of the indicated genotypes imaged for GFP (A,D,G,J,M,P,S) fluorescence and β-Gal (B,E,H,K,N,Q,T) and Ci¹⁵⁵ (C,F,I,L,O,R,U) immunofluorescence. Dashed lines indicate the division between the anterior and posterior compartments, dotted lines indicate regions of high β-Gal and Ci¹⁵⁵ expression within and anterior to the DVS region (N,O, respectively). The boxed regions (P-R) indicate a region harbouring three clones overexpressing β-Gal and Ci¹⁵⁵ in the anterior compartment, which are enlarged in (S-U). (V) Boxplots of quantification of the average β-Gal pixel intensity of non-GFP masked off images (not shown). n = >5 for each genotype, s.e.m indicated. Statistical analysis by one-way ANOVA and Tukey’s multiple comparison tests, which revealed all comparisons to be statistically significant, except those indicated as non-significant (ns). (W) Potential model to explain the effects observed in the posterior EA disc. The double-headed arrow indicates a physical interaction, the single-headed arrow a positive regulatory action and the round-headed arrow a negative regulatory action. Scale bar = 50μm.</p

<i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit increased <i>hh-lacZ</i>-associated β-Gal expression within the posterior compartment and DVS-region.

<p>Confocal image sections of <i>FRT82B</i> control (A-E, L-N)) and <i>hyd</i>^{<i>k7</i>.<i>19</i>} (F-J, O-T) EA discs imaged for direct GFP fluorescence (A,F,N,Q,R), β-Gal (B,C,G,H,L-Q,S) and Ci¹⁵⁵ immunofluorescence (D,E,I,J,M,P,T). (A-K) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibited increased β-Gal expression (H) relative to <i>FRT82B</i> controls (C). Non-clonal regions (GFP—ve regions) were ‘masked off’ to help visualise β-Gal and Ci¹⁵⁵ expression only within GFP-positive clones (C,H and E,J, respectively). Yellow dotted lines indicate the division between anterior and posterior compartment (B—E and G-J). Dashed yellow lines indicate regions of high <i>hh</i> expression (H) and corresponding low Ci¹⁵⁵ expression (E). (K) Quantification of the β-Gal average pixel intensity of the masked off images. n = 5, s.e.m and indicated p value determined by Student’s t-test. Scale bars = 50μm. (L-Q) <i>hyd</i>^{<i>k7</i>.<i>19</i>} DVS regions exhibited abnormal β-Gal (O) and Ci¹⁵⁵ (P) expression. Dashed lines indicate high Ci¹⁵⁵ DVS expression (M,P), which are overlaid onto the other panels. The dotted line marks the anterior front of high β-Gal expression (L), which is overlaid on (M,N). (R-T) Two GFP positive <i>hyd</i>^{<i>k7</i>.<i>19</i>} clones (R), located in the posterior compartment clearly overexpressed β-Gal (S). Of those clones, only one (R yellow dashed line, which is overlaid onto S,T) also harboured increased Ci¹⁵⁵ expression (T). A specific clonal subregion (T, dotted line) with the clone coincided with low β-Gal expression (S, dotted line). Scale bars = 10μm.</p

Sgg and Hyd genetically interact to govern animal viability and head and wing development.

<p>(A-J) Sgg perturbation modifies the <i>hyd</i>^{<i>k7</i>.<i>19</i>} head phenotype. (A-D) Brightfield images of adult <i>Drosophila</i> heads of the indicated genotypes shown either ‘head on’ (upper panels) or ‘side on’ (lower panels). Both gain (C) and loss (D) of <i>sgg</i> function appeared to rescue the <i>hyd</i>^{<i>k7</i>.<i>19</i>} phenotype. Boxplots indicating head width (E, n = ≥8 for each genotype) and counts of eye scars (F, n = ≥8 for each genotype) of the indicated genotypes, with statistical analysis by one-way ANOVA (E) and Fishers exact test (F) revealed statistical significance (asterisks). (G-J) Representative GFP fluorescent signals in adult <i>Drosophila</i> heads of the indicated genotypes revealed only <i>hyd</i>^{<i>k7</i>.<i>19</i>}<i>+sgg</i>^<i>S9A</i> animals lack a GFP signal (n = ≥4 for each genotype). Scale bars = 175μm. (K-P) <i>hyd</i>^<i>WT</i> overexpression promotes the <i>sgg</i>^<i>S9A</i>-mediated wing phenotype. (K-N) Brightfield images of adult <i>Drosophila</i> wings showing (K) normal, (L) mildly deformed, (M) severely deformed and (N) wing-to-notum phenotypes. (O) Percentage of adult wing phenotypes of <i>vg-GAL4</i> flies expressing the indicated transgenes, revealing that the <i>hyd</i>^<i>WT</i> transgene enhanced, and the <i>hyd</i>^<i>C>A</i> transgene suppressed the severity of the <i>sgg</i>^<i>S9A</i> wing defects (n ≥12 for each genotype). (P) Model showing the genetic interaction between <i>sgg</i>^<i>S9A</i> and <i>hyd</i> UAS-transgenes with respect to the wing-to-notum phenotype. Arrows indicate promotion and blockhead arrows inhibition. (Q-R) <i>hyd</i>^<i>WT</i> overexpression rescues <i>sgg</i>^<i>RNAi</i>-mediated embryonic lethality. Percentage viability of <i>sca-GAL4</i> flies expressing the indicated transgenes revealed a >95% rescue of embryonic lethality upon co-expression with the <i>UAS-hyd</i>^<i>WT</i>, but not <i>UAS</i>-<i>hyd</i>^<i>C>A</i>, transgene (16 individual crosses per genotype). (R) Model showing the genetic interaction between <i>sgg</i> and <i>hyd</i> UAS-transgenes. Arrows indicate promotion, blockhead arrows inhibition and dotted blockhead arrow weak inhibition. Scale bar = 250μm.</p

Hyd binds the Hedgehog pathway’s key transcriptional effector Ci¹⁵⁵ and the Ci-regulatory kinase Sgg.

<p>Co-immunoprecipitation (A,D) and affinity-purification (B,C) studies with the indicated affinity reagents were examined by SDS-PAGE and Western blotting with the indicated antibodies. (A) <i>Drosophila</i> CL8 cells were lysed and incubated with either Hyd or control IgG antibodies and affinity purified by Protein G beads. An arrow indicates the position of the expected size band and an asterisk indicates the presence of an uncharacterised faster migrating Hyd species. (B) Mammalian HEK293 cells were transfected with the indicated constructs and lysates underwent Streptactin-mediated purification (Strp) to purify Haemagglutinin-Streptactin-EDD (HS-EDD) and detect co-purified Myc-GLI2. (C) <i>Drosophila</i> S2 cells were transfected with either <i>HS-hyd or HS-vector</i> control, lysed and then incubated with Streptactin-affinity resin. Control and Hyd-coated beads were then incubated with Sgg-FLAG expressing S2 lysate and, following washing, analysed for bound Sgg-FLAG. Only the HS-Hyd beads purified FLAG-Sgg. (D) <i>Drosophila</i> S2 cells were co-transfected with the indicated <i>hyd</i> mutant and <i>sgg</i>-<i>FLAG</i> constructs and FLAG-affinity purified complexes were analysed with the indicated antibodies.</p

<i>hyd</i>^{<i>k7</i>.<i>19</i>} clones exhibit distinct patterns of Ci¹⁵⁵ expression.

<p>Confocal image sections of <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs imaged for direct GFP fluorescence (B,E,F,G,I,J,M,O) Ci¹⁵⁵ immunofluorescence (C,D,F,H,I,L,N,O) and DAPI (A,D,E,K,M,N). (A-F) <i>hyd</i>^{<i>k7</i>.<i>19</i>} discs exhibited curved arrays of nuclei (A, dashed line) that were reflected in the Ci¹⁵⁵ DVS (C, dashed line). (G-I) Posterior <i>hyd</i>^{<i>k7</i>.<i>19</i>} clones near the DVS exhibited increased Ci¹⁵⁵ expression (H, dashed outline). (J-O) Anterior <i>hyd</i>^{<i>k7</i>.<i>19</i>} clones near the DVS exhibited decreased Ci¹⁵⁵ expression (L, low Ci¹⁵⁵ marked by dashed lines, which are overlaid onto J,K). Scale bars = 10μm.</p

<i>hyd</i>^{<i>K7</i>.<i>19</i>} is defective in HECT E3 function and causes abnormal head development.

<p>(A) Schematic representation of the full length Hyd protein containing the Ubiquitin Association Domain (UBA), Regulator of Chromatin Condensation-like (RCC), Ubiquitin-Protein Ligase E3 Component N-Recognin (UBR) domain, Poly(A)-Binding Protein C-Terminal (PABC) and Homologous to the E6AP Carboxyl Terminus (HECT) domains and the potential protein products encoded by <i>hyd</i>^{<i>k7</i>.<i>19</i>} and <i>hyd</i>^<i>15</i>. In comparison to control heads (B-D), <i>hyd</i>^{<i>k7</i>.<i>19</i>} flies (E-G) exhibited disruption of the adult eye and increased head-capsule area. Co-expression of the <i>hyd</i>^<i>WT</i> (K-M), but not <i>hyd</i>^<i>C>A</i> (H-J), transgene suppressed the <i>hyd</i>^{<i>k7</i>.<i>19</i>} phenotype. Scale bar = 200μm. (O) <i>hyd</i>^{<i>K3</i>.<i>5</i>} flies exhibit eye tissue outgrowths that are not present in <i>hyd</i>^{<i>k7</i>.<i>19</i>} heads. (P) Quantification of the head capsule area of the indicated genotype. % values are normalised to control. n = >10 of each genotype. s.e.m and indicated p value determined by Student’s t-test.</p

<i>hyd</i>^{<i>K7</i>.<i>19</i>} EA discs exhibit aberrant Ci¹⁵⁵ expression patterns and morphogenetic furrow-associated features.

<p>(A-D) Deconvolved widefield and confocal image (F-Q) sections of control <i>FRT82B</i> (A,B, F-K)) and <i>hyd</i>^{<i>k7</i>.<i>19</i>} (C,D, L-Q) EA discs imaged for direct GFP fluorescence (A,C,G,K), Ci¹⁵⁵ immunofluorescence (B,D,H,I,K) and DAPI (A,B,C,E,F,G). (A-D) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit abnormal Ci¹⁵⁵ expression patterns. A “Union Jack” lookup table was applied to Ci¹⁵⁵ images to visualise low (blue), medium (white) and high (red) intensity levels and arrows marks the presumed Ci¹⁵⁵ DVS / morphogenetic furrow and an asterisk indicates increased Ci¹⁵⁵ staining as a result of the tissue folding over in itself (B). (D) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibited ectopic Ci¹⁵⁵ expression in the posterior compartment (E, marked by a dashed yellow line, which is also overlaid onto C). (E) Quantification of the area of medium-to-high Ci¹⁵⁵ signal in control and <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs. n = 5, s.e.m and indicated p value determined by Student’s t-test. (F-Q) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit abnormal markers of the morphogenetic furrow. Control FRT82B EA discs exhibited normal nuclei distribution (F) and DVS Ci¹⁵⁵ expression (H), while <i>hyd</i>^{<i>k7</i>.<i>19</i>} discs exhibited irregular patterns (L,N). (F-H) Dashed lines indicated the DVS’s associated high anterior and low posterior Ci¹⁵⁵ expression margins (H), which is overlaid onto (F,G). (L-N) A region of low Ci¹⁵⁵ expression flanked by two DVS-like regions of high Ci¹⁵⁵ expression is marked by a dashed outline (N), which is overlaid onto (L,M). Arrows mark high Ci¹⁵⁵ DVS (H), or DVS-like (N), signals. Scales bars (A-D) 50μm and (F-Q) 10μm.</p