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1 Department of Molecular Microbiology and Biotechnology, and 2 Department Plant Sciences, Tel Aviv University, Tel Aviv, Israel
3 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
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Abstract |
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Introduction |
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Genetic and molecular characterization of the CSN in diverse organisms indicate that it is involved in the regulation of a variety of signaling and developmental processes, including DNA repair, cell cycle regulation, MAPK cascade, embryogenesis, axonal guidance, immune response and hormonal signaling. The details of the mechanism underlying the involvement of the CSN are slowly being revealed, based mainly on identification of CSN-interacting proteins. These studies, and characterization of a key biochemical activity of the CSN, namely deneddylation, indicate that the CSN is involved in regulation of protein degradation.
The CSN interacts with E3–ubiquitin ligases, removing Nedd8, a ubiquitin-like modification, from cullin-based E3's (Lyapina et al. 2001; Wolf et al. 2003) thereby regulating ligase activity. This deneddylation activity resides in the JAMM/MPN+ domain of CSN5, but is dependent on the integrity of the entire complex (Cope et al. 2002). The CSN also interacts with kinases and mediates phosphorylation of ubiquitin–proteasome pathway substrates, and as a consequence alters their stability and sub-cellular localization (reviewed in Harari-Steinberg & Chamovitz 2004).
The exact form of a functional CSN is not clear. Indeed, the need for an eight-subunit complex to carry out deneddylation is not clear. While enzymatic activity resides only in CSN5, this activity is dependent on complex integrity. However, the CSN has functions beyond deneddylation. For example, studies in Arabidopsis have shown that a partial deletion of CSN1 allows complex assembly and CSN5-mediated neddylation, yet still leads to certain phenotypes consistent with a loss of CSN function (Wang et al. 2002). In Arabidopsis, while some of the eight subunits, such as CSN subunit 8 (CSN8), are present only in approximately 500 kDa core complex, some, such as CSN5 and CSN7, are present also in forms independent of the eight-subunit holo-complex (Kwok et al. 1998; Karniol et al. 1999). Several studies have suggested various types of equilibria between these different forms of subunits (Tsuge et al. 2001; Oron et al. 2002; Fukumoto et al. 2005). This dynamic nature has consequences for experiment interpretation. It is still unclear in many cases which of the activities and phenotypes attributed to CSN reside in the complex as a whole, and which reside in the individual subunits (or other complexed forms of the subunits).
We use Drosophila as a model for systematic examination of the roles of the CSN in animals using molecular genetic tools. The Drosophila CSN is highly similar to both mammalian and plant CSNs (Freilich et al. 1999). Genetic analysis of mutants in csn4 and csn5 indicated that the CSN is indispensable for fly development. For example, it is required for oogenesis, embryogenesis, pathfinding by optic axons and differentiation of eye ommatidia, for molting as well as for proper function of the hematopoietic/immune system (Oron et al. 2002; Suh et al. 2002; Harari-Steinberg et al. 2007). Interestingly, certain of these phenotypes are specific for mutants in only one subunit. For example, csn5null mutants die later in the larval stage than csn4null mutants and display hematopoietic/immune defects, while csn4null mutants exhibit primarily molting defects. These differences may in part be attributed to the observation that csn4 mutants lack the CSN core complex, whereas csn5 mutants do not (Oron et al. 2002).
We are especially interested in elucidating the roles of individual subunits in CSN function. In the present study, we describe the generation and characterization of null mutants in a third Drosophila CSN subunit, CSN8. Previous work in Arabidopsis showed that CSN8 is an obligatory subunit of the CSN (Kwok et al. 1998), and in mice CSN8 is expressed throughout early embryonic development, suggesting a key developmental role (Lykke-Andersen & Wei 2003). We observed that CSN8 is found exclusively as part of the CSN core complex, where it has an essential structural function. Assembly of the complex is defective in the csn8null mutants and this affects CSN function since deneddylation of Cullin1 in these mutants is defective. The csn8null mutants are larval lethal, with some reaching prepupation. The mutant larvae have defects in molting from the 2nd to the 3rd instar, and they harbor melanotic capsules in their hemolymph. Mosaic analysis indicated that CSN8 is required for normal development of the wings, eyes and thoracic structures, as well as for oogenesis. Taken together our results indicate that CSN8 is essential for normal function of the CSN and demonstrate its key role in animal development.
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Results |
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The predicted gene encoding the Drosophila CSN8 subunit resides between the genes RpS13, encoding a ribosomal protein, and Pp2A, encoding protein phosphatase 2A. The close proximity to Pp2A, and a report on an EST (SD12355) common to csn8 and Pp2A (Mayer-Jaekel et al. 1992), suggested that csn8 may be transcribed as a dicistronic mRNA with Pp2A (Freilich et al. 1999), in addition to csn8-specific and Pp2A-specific transcripts.
RT-PCR using primers corresponding to exons of the predicted csn8 demonstrated that csn8 is transcribed at all stages of the Drosophila life cycle (Fig. 1A). To examine whether this is in fact a dicistronic csn8–Pp2A transcript, we repeated the RT-PCR, using a Pp2A-specific primer with a csn8-specific primer. No band was amplified at any of the developmental stages examined (data not shown). We conclude that csn8 is transcribed independently of Pp2A and that a dicistronic transcript is unlikely or is present at undetectable levels. These conclusions are supported by genetic analysis described later.
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CSN8 is detected exclusively as part of the CSN complex
Previously studied CSN subunits have been detected in both CSN-dependent (i.e. holo complex) and CSN-independent (e.g. smaller complexes or monomer) forms (Freilich et al. 1999; Oron et al. 2002). To assess if CSN8 is also found in different biochemical conformations, total soluble protein from 3rd instar wild-type (wt) larvae was fractionated by gel filtration chromatography, and probed with anti-CSN8 antibodies. As shown in Fig. 1C, CSN8 is detected exclusively in the higher fractions characteristic of the approximately 500 kDa CSN holo-complex. Thus, as opposed to Drosophila CSN4, 5 and 7, CSN8 appears to function primarily, if not exclusively, in the context of the entire complex.
Generation of csn8 mutants
To gain insight into the contribution of CSN8 to CSN function in the context of the intact organism, we generated mutants in the csn8 gene. A strain carrying a P transposable element inserted between RpS13 and csn8 (Fig. 2A) was obtained. This insert is homozygous viable displaying no obvious mutant phenotype. We mobilized this P-element by hybrid dysgenesis and searched for lethal imprecise excisions that delete flanking chromosomal sequences (Fig. 2B) (see Experimental procedures).
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Molecular analysis showed that in both csn840-1 and csn850-2, a deletion extends from the site of the P insertion into csn8, and that in both mutants the deletion is confined to the csn8 gene: In csn840-1 the deletion removes about half of the coding region of csn8 whereas in csn850-2 it deletes the entire coding region up to the beginning of the 3'UTR of the gene (Fig. 2B).
To verify that csn840-1 and csn850-2 are true null csn8 mutants, immunoblot analysis was performed on total protein extracted from 3rd instar larvae homozygous for each of these mutations, as well as for the hetero-allelic combination csn850-2/csn8SH1829. As expected, no detectable level of CSN8 were detected in any of these mutant genotypes (Fig. 2C).
csn8null mutants display both molting defects and melanotic tumors
Null mutations in each Arabidopsis CSN subunit lead to essentially identical phenotypes (Serino & Deng 2003). However, our previous study of the csn4null and csn5null mutants showed that each displayed both shared and unique phenotypes, consistent with each subunit having a shared function as a member of the CSN holo-complex, but also unique functions (Oron et al. 2002, 2007). Similar to mutants in csn4 and csn5, the csn8 mutants are lethal, none reaching the adult stage. Lethality is evident from the 3rd larval instar on, at which point there is variability in the lethal stage, with the hetero-allelic combination csn850-2/csn8SH1829 being somewhat less severe than csn850-2-homozygotes (Fig. 3). As the hetero-allelle is deneddylase null (see Fig. 8 below), and as csn8SH1829 homozygote larvae are also less robust than the hetero-allelic larvae (not shown), the increased robustness of the hetero-allelic larvae is likely a result of genetic background effects. While the majority of mutant larvae never reach the pupal stage (similar to both csn4null and csn5null mutants) and are markedly smaller than their normal heterozygote siblings (similar to csn4null mutants) (Fig. 4A), a subpopulation of larvae maintain normal body size, but have a 1- to 2-day delay in the transition to the prepupa stage, at which point they arrest (Fig. 3).
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Since the melanotic capsules phenotype is reminiscent of phenotypes reported for immune response and hematopoiesis mutants, we hypothesized that CSN8, like CSN5, is involved in the regulation of immune signaling in Drosophila. To test this, we examined the resistance of csn850-2/csn8SH1829 larvae to bacterial immune challenge (Table 1). Wild-type (wt) and csn850-2/csn8SH1829 3rd instar larvae, 72 h after egg deposition (AED), a developmental stage when wt and mutants are still indistinguishable, were bacterially challenged by pricking them with a needle dipped in a bacterial cocktail and their viability was monitored 24 h later, relative to viability of larvae pricked with a sterile needle. While both mutant and wt showed reduced survival upon pricking with a sterile needle, the bacterial challenge had no effect on survival of wt larvae, but caused 32% mortality among mutant larvae. As an additional control, the experiment was also carried out on heterozygous csn850-2/+ or +/csn8SH1829 siblings of the mutants. These siblings also showed no sensitivity to the bacterial challenge. This indicates that csn850-2/csn8SH1829 larvae are hypersensitive to bacterial immune-challenge, displaying a risk value of 1.48.
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Immunoblot analysis on unfertilized wt eggs indicated that CSN8 is maternally contributed to the embryo (Fig. 2). To determine the persistence of maternally contributed CSN8, immunoblots were carried out on proteins from staged mutant larvae. As seen in Fig. 5, the csn850-2 1st larval instar maintains an appreciable level of the CSN8 protein. The maternally contributed CSN8 gradually decreases from the 1st to the beginning of the 2nd larval instar, from which point on, no CSN8 is detectable in the mutants.
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This was confirmed by dissection of ovaries from the females carrying the germ-line clones (Fig. 6). As expected, ovaries of positive control females (heat-shocked hs-FLP; FRT csn8+/FRT ovoD) were fully developed, whereas oogenesis in ovaries dissected from negative control females, carrying the OVOD mutation, was found to be arrested at stage 4. Oogenesis in females carrying germ-line clones of csn850-2 (hs-FLP; FRT csn850-2/FRT ovoD) arrested by stage 7, with no mature egg chambers detected. These results indicate that CSN8, and by association the CSN holo-complex, are necessary for oogenesis.
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As the above analysis indicated that CSN8 is necessary for germ-line and larval development, we examined if it is required for post-larval development. To test this possibility, csn8null mitotic clones were generated in otherwise normal adult flies by heat-shock induced FLP–FRT. Such clones induced in the larval imaginal discs resulted in disruption of the corresponding adult structures (Fig. 7). For example, eyes containing csn850-2 homozygous clones were small, bulging from the head. The eye surface was rough and lacked the orderly lattice-like arrangement of normal ommatidia. The eyes also contained melanized areas. Other head-capsule structures derived from the eye imaginal disc, such as the vibrissae and the antennae, were also affected.
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To determine if these phenotypes are specific for CSN8 (and thus the entire CSN) or are also dependent on CSN5, similar csn5null mitotic clones were also generated. As seen in Fig. 7J–L, csn5null clones in both eye and wing led to essentially identical phenotypes as the corresponding csn8null clones.
Function and integrity of the CSN complex are disrupted in csn8 mutants
We assessed the effect of the lack of CSN8 on the ability of the CSN to de-neddylate Cul1. Total protein was extracted from csn850-2/csn850-2 and csn850-2/csn8SH1829 3rd instar larvae, and immunoblotted with antibodies against Cul1. Extracts from wt and csn5null homozygous larvae served as controls. In the wt 6 times as much Cul1 was present in non-neddylated form as compared to neddylated Cul1, whereas in the csn5null mutant about twice as much Cul1 was in the neddylated form, reflecting the impaired de-neddylase activity of this mutant. Strikingly, in the two csn8null genotypes examined, only neddylated Cul1 was detected (Fig. 8A). Thus, the loss of CSN8 leads to a complete loss of CSN-dependent de-neddylase activity.
We next sought to assess the contribution of CSN8 to the stability of the CSN holo-complex. The Arabidopsis csn8 mutant shows accumulation of the monomeric form of CSN5, indicating that CSN8 is essential for integrity of the complex (Kwok et al. 1998). To examine if this role of CSN8 is conserved in Drosophila, total proteins from 3rd instar larvae homozygous for the csn850-2 null mutation, as well as from the hetero-allelic combination csn850-2/csn8SH1829, were size fractionated, and the resulting fractions analyzed by immunoblot with anti-CSN5 antibodies. As seen in Fig. 8B, while in the wt CSN5 is detected in two distinct peaks, one around 500 kDa, corresponding to the intact CSN holo-complex, and the other in smaller fractions (approximately < 100 kDa) corresponding to the CSN-independent forms of CSN5 (Oron et al. 2002), in csn850-2/csn8SH1829 larvae, as well as in the individual homozygous null strains, CSN5 is detected only in the small CSN-independent forms. Thus, either assembly or stability of the CSN holo-complex is impaired in the absence of CSN8.
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Discussion |
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Indeed, CSN8 is such a subunit. CSN8 is detected exclusively as part of the approximately 500 kDa holo–CSN complex in Drosophila, similar to CSN8 in both the plant and mammalian CSN complexes (Wei & Deng 1998; Oron et al. 2002). Furthermore, in the absence of CSN8, no holo–CSN complex can be detected. This indicates that CSN8 is necessary for the assembly and/or stability of the holo-complex. Thus, fly CSN8 has a role in maintaining the structural stability of the complex, as found for Arabidopsis CSN8 (Kwok et al. 1998), further demonstrating the evolutionary structural conservation of the CSN.
Interestingly, while CSN8 has a conserved structural role in the complex, it is the least conserved subunit in terms of amino acid identity. Indeed, no CSN8 orthologue is detected in the Neurospora crassa, Schizosaccharomyces pombe or Saccharomyces cerevisiae genome, and their corresponding CSN complex have less than eight subunits (Zhou et al. 2001; Maytal-Kivity et al. 2003). So while CSN8 is clearly essential for CSN stability in metazoans, unicellular organisms can form a functional CSN in its absence.
As such, it is informative to compare the phenotypes of csn8null mutants with those of previously published Drosophila mutants in the CSN. csn8null mutants are lethal, indicating that CSN8, similar to CSN4 and CSN5, is essential for Drosophila development. However, the lethal phase of the csn8null mutants differs from the other mutants in that: (i) they are more variable, with individual larvae arresting at various stages from the 2nd–3rd instar transition [similar to csn4null (Oron et al. 2002)] through to late 3rd instar [similar to csn5null (Oron et al. 2002; Harari-Steinberg et al. 2007)]; and (ii) a small percentage of the csn8null mutants pupariate and arrest as prepupa, whereas none of the other csn mutants reach the prepupal stage. Indeed, while csn5null mutants survive in a prolonged 3rd larval instar, they never show wandering behavior or initiate pupariation (Harari-Steinberg et al. 2007).
The survival of the csn8null mutants through a major part of development may seem at odds with the fundamental roles and conservation of the CSN complex, and specifically, with the early oogenic arrest phenotype of germ-line clones. This extended viability of the mutants can be explained by two non-exclusive hypotheses. First, as CSN8 is essential for CSN complex integrity, the function of individual subunits (such as CSN4, CSN5 or CSN7) outside the complex remain unaffected in the csn8null mutants, as opposed to the csn4null or csn5null mutants, where their respective monomers are also deleted. Thus the earlier lethality of the csn4null and csn5null mutants would arise from the loss of both the CSN complex and one of the monomers, which is not the case for the csn8null mutants. Alternatively, the extended viability of the csn8null mutants can be explained by maternally contributed CSN8. Indeed, CSN8, like CSN4 and 5, is maternally contributed to the egg, and maternal persistence of CSN8 is longer than that of CSN5, which is depleted by late embryogenesis (Oron et al. 2002). The level of maternal CSN8 diminishes gradually until it is completely depleted by the mid-2nd larval instar, at which time its absence likely initiates the derailment which leads later to the developmental arrest. Accordingly, at the cellular level, the absence of either CSN8, 5 or 4 leads to equally severe the germ-line phenotypes (Oron et al. 2002).
The csn8null mutants display two overt phenotypes: double mouth-hooks structures and melanotic capsules. The double mouth-hooks phenotype is common to csn4null and csn5-point mutated hypomorphic mutants (Oron et al. 2002, 2007). This well-studied phenotype reflects a block in the molting process. Such molting arrest is characteristic of mutants affecting signaling through the steroid molting hormone ecdysone (Thummel 1996; Freeman 1999; Davis et al. 2005). Involvement of the CSN in ecdysone signaling in Drosophila was originally proposed after finding that the fly CSN2 (alien) binds EcR in yeast two-hybrid assay and is capable of binding the thyroid hormone receptor in a hormone-sensitive manner (Dressel et al. 1999). More recently, in a genome-wide expression profiling, we found that expression of genes in ecdysone signaling pathway is altered in csn4 and csn5 mutants (Oron et al. 2007). Taken together, these observations suggest that the CSN complex is involved in regulation of insect steroid hormone signaling.
The csn8null mutants that did survive into the 3rd larval instar displayed melanotic capsules floating in their hemolymph. Similar melanotic capsules are characteristic of immune response and hematopoiesis mutants of the fly (reviewed in Minakhina & Steward 2006) and are also characteristic of csn5 mutants (Oron et al. 2002). Indeed, similar to lack of CSN5 (Harari-Steinberg et al. 2007), lack of CSN8 also leads to reduced resistance to bacterial infection, as the mutants are sensitive to bacterial challenge. The phenotypic overlap in immune responses between csn5 and csn8 mutants strongly suggests that this function is mediated by the holo–CSN complex. This is supported by recent work in mice which showed that csn8null mice are immune defective (Menon et al. 2007).
Here we showed that the involvement of the CSN in regulation of cell proliferation and differentiation in Drosophila extends beyond the larval hematopoietic system. Clones of cells lacking CSN8 induced in the wing and eye discs display growth defects. This defect likely reflects a derailment cell cycle. Indeed, many studies have implicated the CSN in cell cycle regulation in general (Gemmill et al. 2005; Richardson & Zundel 2005; Bjorklund et al. 2006; Ullah et al. 2007), and specifically in regulating Cullin-based E3 activity in Drosophila eye development (Ou et al. 2002). While these studies have not usually addressed the question as to whether the CSN–cell cycle connection was dependent on the entire CSN complex or on specific subunits, our results suggest that this may be complex dependent. The genetic tools developed here can be used to further explore this question.
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Experimental procedures |
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Wild-type (wt) (Canton-S) and mutant strains were reared on standard cornmeal-molasses medium at 25 ºC. Experiments were carried out at 25 °C or 18 °C as indicated. Larval staging was performed following a 4-h period of egg laying. Heat-shock induction of mitotic clones was performed by exposing staged larvae twice a day, on days 3 and 4 to 1 h of 37 °C in a water bath after which the larvae were returned to 25 °C. Mobilization of P-element and screening for excision events was as described (Freilich et al. 1999; Oron et al. 2002).
Mutant strains used
Where needed, strains were rebalanced over corresponding GFP-bearing balancer chromosomes to facilitate identification of the desired classes of offspring.
Immune response and statistical analysis
Third instar larvae, 72 h AED, were challenged by pricking with a needle dipped in a cocktail of Escherichia coli and Micrococcus luteus cultures, and their viability was assessed, 24 h later as done previously (Harari-Steinberg 2006). For the survival experiments, two controls were used, Canton-S, and heterozygote siblings of the mutants. Relative risk value shows the ratio of the risk of death relative to exposure in the mutant vs. the wt control group.
Molecular methods
Extraction and analysis of DNA, RNA and proteins were as described previously (Oron et al. 2002; Harari-Steinberg et al. 2007).
PCR primers used for analysis of DNA lesion in the mutants:
PCR primers used for RT-PCR analysis:
Antibody production
For production of antibodies against the Drosophila CSN8 (herein termed 
-dmCSN8) a full-length cDNA clone of csn8 was obtained from the Berkeley Drosophila Genome Project (EST: SD12355). EcoRI and XhoI restriction sites were added at the 5' and 3' ends, respectively, and the construct was cloned into pET-28a (Novagen, Madison, WI) to over-produce His-tagged CSN8 in E. coli Tuner(DEB)CodonPlus bacteria. Upon 100 000 g centrifugation the CSN8–His fusion protein was found in the soluble fraction. It was purified on Ni-column, and was used to immunize rabbits (AniLab, Rehovot, Israel).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: dsegal{at}post.tau.ac.il
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References |
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Received: 30 August 2007
Accepted: 20 November 2007
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