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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02743
Research Article |
Universität Hohenheim, Institut für Genetik (240), Garbenstr. 30, 70599 Stuttgart, Germany
* Author for correspondence (e-mail: preiss{at}uni-hohenheim.de)
Accepted 17 October 2005
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Summary |
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Key words: Drosophila, Hairless, Notch regulation, Pros26.4, Protein stability
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; Voges et al., 1999; Glickman and Ciechanover, 2002). The proteasome consists of a 20S central core that confers proteolytic activity, whereas the 19S regulatory caps are involved in substrate recognition and unfolding. The cap can be divided into a lid and a base, and contains six different AAA-ATPases displaying reverse chaperone activity. ATPase activity is required at several steps of protein degradation, e.g. for proteasome assembly and for effective substrate recognition and unfolding. One of the six AAA-ATPases, Rpt5 (or S6'), has been shown to contact poly-ubiquitin chains on substrate proteins only, however, when in a complex with the other five AAA-ATPases (Lam et al., 2002). This suggests an intimate interaction amongst the six AAA-ATPases and, furthermore, a general involvement of the base in substrate recognition. Thereby, ATPase activity may be contributed from any of these proteins (Rubin et al., 1998; Lam et al., 2002). The AAA-ATPase Rpt2 (or S4) stands out in some respects. It belongs to the four proteins of the cap most conserved in the evolution from yeast to human (Hölzl et al., 2000; Wollenberg and Swaffield, 2001). It is indispensable for yeast vitality and is highly susceptible to mutation (Schnall et al., 1994; Rubin et al., 1998). It was shown to enhance the peptidase activity of the proteasome, presumably by gating the central core of the proteasome's hydrolytic 20S unit (Rubin et al., 1998; Kohler et al., 2001). We have isolated the orthologous Pros26.4 gene from Drosophila melanogaster in a yeast two-hybrid screen for interaction partners of Hairless. Hairless plays a major role as an antagonist in the Notch signalling pathway in Drosophila.
In higher eukaryotes, the Notch signalling pathway mediates communication of neighbouring cells (for reviews, see Artavanis-Tsakonas et al., 1999; Schweisguth, 2004). Cell surface signals from one cell activate the Notch receptor in the adjacent cell. The receptor is subsequently cleaved and the intracellular domain of Notch, NICD, is released. NICD acts as a transcriptional co-activator of CSL-type DNA-binding proteins, named Suppressor of Hairless [Su(H)] in Drosophila. Apparently, transcriptional activation is accompanied by proteasome-mediated degradation of NICD as a means of negative regulation of this pathway (Schweisguth, 1999; Fryer et al., 2004) (reviewed in Schweisguth, 2004). Independent of this, Notch target genes are subjected to transcriptional repression by a repressor complex involving Hairless (H) and the general co-repressors CtBP and Groucho in Drosophila (Morel et al., 2001; Barolo et al., 2002; Nagel et al., 2005). To investigate the role of Hairless in more detail, a yeast two-hybrid screen for interaction partners of Hairless was carried out (Marquart, J. Identifizierung von Interaktionspartnern des Drosophila-Hairless Proteins und Charakterisierung seiner bindungsrelevanten Bereiche. PhD Thesis, Universität Hohenheim, 1999). This led to the identification of the Pros26.4 gene, which encodes the Rpt2 orthologue in Drosophila (Hölzl et al., 2000). In accordance with its function in the proteasome, Pros26.4 encodes a general, essential factor during fly development. Knock-down of Pros26.4 activity by RNA interference (RNAi) caused lethality and cell degeneration in accordance with its essential cellular role. At the same time Pros26.4 depletion resulted in a specific stabilization of Hairless but neither of Su(H) nor Notch proteins. The Hairless-Pros26.4 interaction might thus reflect a novel mechanism of positive regulation of the Notch signalling pathway.
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Expression of Pros26.4 during Drosophila development
Molecular interactions are relevant only when the respective proteins are expressed at the same time within the same tissues during development. We thus analysed the expression patterns of Pros26.4 mRNA and protein. Pros26.4 mRNA is uniformly distributed in early eggs owing to maternal product (Fig. 2A), as has been described before for Hairless transcripts. Later in development, transcripts appeared enriched within the developing nervous system (Fig. 2B-H) thus overlapping the Hairless expression domain (Maier et al., 1992). In third instar larvae, Pros26.4 mRNA levels were higher in dividing cells, e.g. in neuroblasts of the ventral chord or in eye imaginal discs anterior to the morphogenetic furrow (Fig. 2I-L). This pattern is very similar to that of Pros28.1, which encodes an 
-subunit of the central core (Fig. 2M-P; Udvardy, 1993). Pros26.4 protein is ubiquitously distributed in all tissues; it is cytoplasmic and also, albeit to a lesser degree, nuclear (Fig. 3C). During oogenesis the protein is enriched in the germinal vesicle (Fig. 3A,B) as was reported before for the - and ß-subunits of the 20S central core (Adam et al., 2004). The general distribution of both Pros26.4 and Hairless proteins (Maier et al., 1999) indicates extensive overlap in many tissues.
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Depletion of Pros26.4 during Drosophila development results in cellular degeneration and death
Pros26.4 was overexpressed in a variety of different tissues using the Gal4/UAS system (Brand and Perrimon, 1993). Despite a strong expression, the flies developed completely normal without any obvious phenotypes (not shown). Based on the close molecular contacts between the six AAA-ATPases forming a ring at the base of the 19S regulatory cap (Wollenberg and Swafield, 2001; Lam et al., 2002), one might expect that overexpression of any of them disrupts the stoichiometry of the ring and, therefore, proteasome activity. However, this is not the case because Drosophila is insensitive to a large increase in the amount of Pros26.4.
Recently, it was shown in Drosophila S2 cells that reduction of Pros26.4 activity by RNAi affects cell growth rates and results in an increased number of apoptotic cells (Wojcik and DeMartino, 2002). Presumably, Drosophila cells that completely lack Pros26.4 cannot survive because respective mutations in yeast are lethal (Rubin et al., 1998). To study loss of Pros26.4 activity in Drosophila, transgenic flies were constructed containing an inverted Pros26.4 construct (UAS-dsS4) thus allowing tissue-specific RNAi (Nagel et al., 2002). In the animal, ubiquitous knock-down with the da-Gal4 driver caused death at about second larval instar. Likewise, larval or pupal death was observed upon tissue-specific RNAi when using a number of different driver lines (e.g. ptc-Gal4, en-Gal4, sca-Gal4). Apparently, depletion of Pros26.4 caused cell death not only in Drosophila cell lines but also during fly development (see Fig. 4). For example, specific expression of UAS-dsS4 within the eye anlagen using ey-Gal4 allowed us to recover pharate adults from the pupal case that lacked the eyes and most of the head capsule (Fig. 4A,G). Closer inspection of the defects caused by loss of Pros26.4 was possible by using gmr-Gal4, which drives expression later in development within differentiating cells of the eye imaginal disc. In these cases, we found fusions of ommatidia in a posterior to anterior gradient in the adult eye (Fig. 4D, compare with 4A). Frequently, necrotic tissue was attached to the very posterior end of the eye. The underlying defect was revealed in sections, which showed a likewise graded loss of internal retinal tissue (Fig. 4E, compare with 4B), reflecting the respective differentiation wave. Only cells behind the morphogenetic furrow, which sweeps from posterior to anterior over the eye imaginal discs during larval life, express gmr-Gal4 (Wolff and Ready, 1993). Thus, cells furthest anterior were the last to be exposed to RNAi and survived, whereas those more posterior were destroyed. A strong increase in cell death was already observed in third larval instar eye discs (Fig. 4C,F), and could be rescued by simultaneous overexpression of the anti-apoptotic baculoviral p35 protein (Fig. 4H). The defects seen in the adult, however, arose later in development, and only a marginal rescue was caused by the overexpression p35 (not shown). Apparently, interference with Pros26.4 activity occurs with a pronounced time-lag, presumably due to a stable pool of Pros26.4 protein. As a consequence, cells eventually die and the retina degenerates with a late onset.
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). However, Hairless gain-of-function phenotypes were not altered by the concurrent overexpression of Pros26.4 (data not shown). Mutations in Hairless are haplo-insufficient and cause a dominant loss of bristles and wing venation defects. This phenotype was rescued to some extent by simultaneously removing one copy of Pros26.4 by using deficiency Df(3R)mbc-R1: flies heterozygous for HP8 in a wild-type background lacked 12.2 macrochaetae on average (see Maier et al., 2002), whereas in the background of Df(3R)mbc-R1, they lacked only 9.7 bristles on average. This slight amelioration of the Hairless loss-of-function phenotype by concurrent loss of Pros26.4 argues for a negative regulation of Hairless by Pros26.4. One role of Pros26.4 might be, for example, to destabilize the Hairless protein. However, this deficiency removes further genes of unknown function that might influence the Hairless mutant phenotype as well. To investigate whether reduction of Pros26.4 activity by RNAi likewise restored bristle numbers, we overexpressed UAS-dsS4 within proneural clusters, including the bristle precursor cells using the sca-Gal4 driver line. Unfortunately, the animals died at late pupal stage and displayed a reduced number of bristles on their own. This is not unexpected, considering that Pros26.4 is required in cell survival as outlined above. However, it prevented us from investigating the genetic interactions. We therefore decided to study eye development instead.
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; Müller et al., 2005). This gain-of-function phenotype was markedly enhanced by a knock-down of Pros26.4: the eyes became even smaller with a very glossy surface, indicative of strong defects in retina development (Fig. 5D). This is in agreement with the hypothesis that Pros26.4 normally downregulates Hairless: reduction of Pros26.4 activity due to RNAi should therefore enhance phenotypes caused by a surplus of Hairless. By contrast, no effect was seen on the enlarged eyes caused by the overexpression of Su(H) or Notch (Fig. 5E-H). Overexpressing either Su(H) or Notch in combination with dsS4 gave the expected additive phenotype: eyes were still enlarged, but showed a glossy appearance and necrotic tissue caused by the depletion of Pros26.4 (Fig. 5F,H).
Pros26.4 depletion results in a stabilization of Hairless protein but not of Notch or Su(H)
To test the effect of Pros26.4 on protein stability more directly, we assayed levels of Hairless, Su(H) or Notch proteins while reducing Pros26.4 by RNAi. Cell death resulting from such a depletion was partially prevented by driving the Drosophila inhibitor of apoptosis protein DIAP1 (Lisi et al., 2000) simultaneously with the dsS4 construct. Notch, Su(H) or Hairless proteins were induced all over and, with respect to Pros26.4 knock-down, stability of each protein was assayed at specific time points after induction. For this experiment, either Notch, Su(H) or Hairless transgenes were induced from a heat-shock promoter by a 1-hour heat-pulse in animals where dsS4 and/or DIAP1 had been activated along the antero-posterior border of imaginal discs (Fig. 6). After a given time at ambient temperature, wing discs were immuno-stained for the respective proteins. They were compared with controls that had not been subjected to the heat-pulse and in which no effect on protein distribution was seen. As shown in Fig. 6A, Hairless protein was slightly stabilized by overexpression of DIAP1 and very strongly, when Pros26.4 activity was also reduced (Fig. 6B,B'). By contrast, the stability of either Notch or Su(H) proteins remained unchanged (Fig. 6C-F') arguing that Pros26.4 had no influence on these proteins. These data are in agreement with earlier findings that RNAi for Pros26.4 does not result in an immediate failure of proteasome activity (Wojcik and DeMartino, 2002; Boutros et al., 2004). Otherwise, we would have expected stabilization of Notch as well, because this effect was observed with a dominant negative mutation in the proteasomal ß-subunit (Schweisguth, 1999). Rather, the particular stabilization of Hairless hints to a specific regulation of this protein by Pros26.4.
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). In accordance with our observation of a significant time-lag of the Pros26.4 RNAi-effects, the cells initially grew normally before growth rate and cell number decreased significantly.
The 19S regulatory particle is indispensable to the recognition and unfolding of proteins destined for degradation by the 20S core particle. Thus, it is not surprising that interference with its activity results in cell death. In addition, it has been assigned a number of other, completely different, activities: in yeast it was shown that the 19S regulatory particle plays an independent role in nucleotide excision repair of damaged DNA and, moreover, in transcription activation and elongation (reviewed in Muratani and Tansey, 2003). To date it is unknown whether these multiple activities also apply to the fly. If this were the case RNAi with Pros26.4 might interfere with all these processes. Interestingly, impeding the activity of the central core subunits ß6 or ß2 causes superficially very similar phenotypes to depletion of Pros26.4 (Belote and Fortier, 2002). The authors mis-expressed respective dominant negative mutants DTS5 and DTS7 in the photoreceptor cells. They observed necrosis after a noticeable time-lag but did not analyse the underlying defects. The stunning resemblance of phenotypes strongly suggests that, above all, RNAi of Pros26.4 interferes with proteasome activity proper. Apparently, the function of Pros26.4 within the proteasome degradation machinery is the most dose-sensitive, such that reduction in activity causes instant and obvious defects. By means of the tissue-specific RNAi assay we can now identify and further analyse possible roles of Pros26.4 in Drosophila, e.g. in gene transcription or nucleotide-excision repair.
The ubiquitin-proteasome system and the regulation of Notch-dependent transcription
In the last few years, an intimate relationship between the proteasome and the regulation of gene expression has been established (for a review, see Muratani and Tansey, 2003). In principle, the proteasome might control transcriptional regulators at the level of availability (i.e. their translocation into the nucleus or into nuclear bodies) or at the level of activity (i.e. by delimiting co-activators or alike) and finally at the level of abundance. Indeed, it has been reported that Notch-mediated gene activation is negatively regulated by targeting the intracellular domain of Notch for proteasomal destruction (Schweisguth, 1999; Gupta-Rossi et al., 2001). Transcription-coupled proteolysis seems common to unstable transcriptional activators, in which trans-activation domains and degradation signals often overlap. Thereby, activation of the transcription machinery is directly linked to the degradation of the trans-activator, a process coined the `black widow' model (Tansey, 2001). In the process of activation, the transcriptional regulator is marked for degradation. Indeed, many transcriptional regulators are phosphorylated by components of the basal transcription machinery. This renders them susceptible to ubiquitylation and degradation by the proteasome (Muratani and Tansey, 2003). In case of Notch signalling the process has been analysed in detail (Fryer et al., 2004). The transcription-activation complex affecting Notch signals includes the intracellular domain of Notch, a CSL-type DNA-binding protein and also the co-activator Mastermind (Mam). Mam is essential for transcription initiation and promotes hyperphosphorylation of Notch by coupling to the CycC:Cdk8 kinase. This results in PEST-dependent degradation of active Notch (Fryer et al., 2004).
Besides coupling transcriptional activation to destruction, a non-proteolytic role for the 19S regulatory particle has been stated in transcriptional activation and elongation. Notably, the proteasomal AAA-ATPases Rpt6/Sug1 and Rpt4/Sug2 are located at promoters where they direct chromatin remodelling (Gonzalez et al., 2002; Muratani and Tansey, 2003; Ezhkova and Tansey, 2004). They are recruited by mono-ubiquitylated histone H2B, which participates in the activation of transcription and relief of telomeric silencing in yeast. H2B ubiquitylation is mediated by the Rad6 complex, which includes the E3 ubiquitin ligase Bre1. It is a first and mandatory step for subsequent histone H3 methylation at lysine residues 4 and 79, which also depends on the presence of Rpt6/Sug1 and Rpt4/Sug2 (Muratani and Tansey, 2003; Wood et al., 2003; Ezhkova and Tansey, 2004). It has not yet been studied in detail, whether similar mechanisms apply to Drosophila as well. However, very recently it has been shown that a mutation in Drosophila Bre1 particularly affects Notch target gene expression (Bray et al., 2005), arguing that, in Drosophila, Notch target genes are particularly susceptible to the effects of histone H2B ubiquitylation. Albeit all six AAA-ATPases quickly associate with active promoters in yeast, there is little evidence for a specific role of Rpt2/S4 in transcriptional regulation (Gonzalez et al., 2002). In this case, knock-down of Pros26.4 by RNAi would result in a downregulation of Notch target genes, similar to mutations in Bre1.
Our results demonstrate that Pros26.4 positively influences Notch signalling by specifically destabilizing the Notch antagonist Hairless. Our data reveal a remarkable, Pros26.4-dependent preference of the proteasome for Hairless compared with other potential substrates such as Notch. We propose that this is based on a direct molecular interaction of Pros26.4 and Hairless. It is conceivable that protein-protein interactions of proteasomal AAA-ATPase subunits with candidate substrates occur independently of ubiquitylation, which regulates proteasomal access. Hairless interferes with Notch activity in two ways. On one hand, it interferes with the assembly of the transcription-activator complex by competing for CSL/Su(H) and on the other hand, it forms, directly on Notch-responsive promoters, a repressor complex together with Su(H), and the co-repressors Groucho and CtBP (Morel et al., 2001; Barolo et al., 2002; Nagel et al., 2005). Thus, by decreasing the availability of Hairless protein, Pros26.4 promotes Notch target gene activation.
In conclusion, transcriptional regulation of Notch target genes is influenced in several ways by the ubiquitin-proteasome system. First, activated Notch undergoes proteasomal destruction in a typical transcriptional activation-destruction process. Second, Notch target genes are particularly susceptible to ubiquitin-dependent chromatin activation. Third, by means of Hairless destabilization, the AAA-ATPase Pros26.4 promotes Notch signalling.
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) with the bait construct pEG-FLH (Maier et al., 1997) according to standard protocols (Fields and Song, 1989). After three rounds of verification, 57 clones were tested positive. Five different genes were isolated; the vast majority of clones (41%) represented Pros26.4 (pJG7-9). In comparison to the published DNA sequence, clone pJG7-9 carried a 39-nucleotide gap at position 137 and contained a 21-nucleotide leader sequence but retained an open reading frame and expressed a fusion protein of the expected size. A full-length Pros26.4 cDNA clone (ICRFp520D102) was obtained from RZPD (Berlin) and used to amplify the Pros26.4 coding sequence with forward primer CAA GCA CCA AGA ATT CAT GGG ACA AA and reverse primer TAT ATC TTC GCT CGA GTT AAT AAA TAA. To generate pJG-S4, the amplified sequence was digested with EcoRI and XhoI, and cloned into likewise linearized pJG4-5 vector (Gyuris et al., 1993). In subsequent qualitative and quantitative yeast two-hybrid assays, pJG-S4 was indistinguishable from pJG7-9. Clones were sequence verified and protein expression in yeast cells was tested in western blots with antibodies directed against HA (Roche Ltd) and LexA (Clontech).
Analysis of protein-protein interactions
Co-immunoprecipitations were performed on protein extracts of approximately 500 wild-type embryos. For immunoprecipitation, we used rabbit anti-Hairless A antiserum at 1:250 dilution and for detection rat anti-Hairless A (Maier et al., 1999) and rat anti-S4 antibodies at 1:500 dilution, respectively. Secondary AP-coupled antibodies were diluted 1:200 (Jackson Laboratories). Paired protein interactions were analysed as previously described (Matsuno et al., 1995). For bait constructs pEG-FLH, pEG-C1, pEG-C2, pEG-C3, pEG-CX and pEG-C6, see Maier et al. (Maier et al., 1997). To map the interacting site of Hairless within Pros26.4, a set of five fragments was generated by PCR using Pfu-polymerase (Stratagene) and Pros26.4 full-length cDNA as template. Forward primers carried an EcoRI and reverse primers a XhoI restriction site to allow cloning in respectively linearized pJG vector (Gyuris et al., 1993). Primer sequences are available upon request. Final constructs were sequence verified.
Manipulation of gene activity
Flies were raised under standard conditions; further information on fly lines can be obtained from http://flybase.bio.indiana.edu/. Tissue-specific expression of transgenes was controlled by use of the Gal4/UAS-system (Brand and Perrimon, 1993). Driver lines were da-Gal4, en-Gal4, ey-Gal4, gmr-Gal4, ptc-gal4 and sca-Gal4. The following UAS-lines were used: UAS-DIAP1 (gift of A. Müller, Heinrich-Heine-Universität, Düsseldorf, Germany), UAS-GFP, UAS-NICD (Go et al., 1998), UAS-Su(H) and UAS-H (Nagel et al., 2000). Heat-inducible lines were hsH, hsSu(H) and hsNICD (Lieber et al., 1993; Schweisguth and Posakony, 1994; Maier et al., 1997). Df(3R)mbc-R1 takes out 95A5/7 to 95D6/11; Pros26.4 is located at 95C and thus contained within the deletion. UAS-S4 transgenic flies were generated with a construct containing the complete open reading frame of Pros26.4 shuttled from pEG as an EcoRI-XhoI fragment into pUAST. Tissue-specific RNAi was induced by overexpression of UAS-dsS4, which was cloned as outlined before (Nagel et al., 2002): a DNA segment covering codons 90 to 250 was PCR amplified with primers containing sites for BamHI (5') CCA GGA TCC GAA GAA CGA GGA GGA GCG CTC TAA and Kpn I (3') CTC GGT ACC TAC AAC GCG CAG GAA AGT GGC C, cloned into pHIBS and shuttled into pUAST, respectively. Transformant flies were generated according to standard protocols. For the genetic interaction studies, the following fly lines were constructed by standard recombination techniques: gmr-Gal4 UAS-H2nd / CyO (Müller et al., 2005); UAS-DIAP1, gmr-Gal4; UAS-DIAP1, UAS-dsS4; UAS-NICD UAS-dsS4; UAS-Su(H) UAS-dsS4 / TM3Sb. Crosses were carried out at 18°C or 25°C as specified and the offspring was compared with the siblings or with control crosses with UAS-GFP lines. Pictures of adult flies were taken with a Leica Wild M3C dissecting microscope equipped with a Pixera digital camera. Scanning-electron micrographs were taken with a Joel JSM35 microscope. For the protein stability experiments, fly stocks of the following genotypes were constructed: UAS-DIAP1, ptc-Gal4; UAS-dsS4 hsNICD; UAS-dsS4 hsH3rd and UAS-dsS4 hsSu(H). Crosses were reared at 25°C. Then a single 1-hour heat-pulse at 37°C was given to wandering third instar larvae, which were aged for 1, 6 or 12 hours at room temperature, before histochemical analysis; they were compared with unshocked control animals.
Immuno-histochemistry
Polyclonal antibodies directed against Pros26.4 and DIAP1 were raised in rats and rabbits immunized with GST- and MBP-fusion proteins, respectively (PINEDA ABservice Berlin). Other primary antibodies were directed against Hairless (Maier et al., 1999), Su(H) (Schweisguth and Posakony, 1994) and Notch (Fehon et al., 1991). The latter was obtained from DSHB (University of Iowa under the auspices of the National Institute of Child Health and Human Development). Secondary antibodies (1:200) coupled to DTAF or Cy3 were purchased from Jackson Laboratory (Dianova). Samples were mounted in Vectashield (Vector Lab) and analysed on a Zeiss Axioskop or a Zeiss Axiophot linked to a Bio-Rad MRC1024 and a Zeiss LSM 510 confocal microscope, respectively. Pictures were assembled using CorelDraw software.
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S; 885 aa); C3, deletion of acidic domain (867 aa); CX, internal deletion within the C-terminal third of the Hairless protein (751 aa); C6, C-terminal truncation of 15 amino acids including the CtBP-binding domain (H
