Genomic analysis of COP9 signalosome function in Drosophila melanogaster reveals a role in temporal regulation of gene expression

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Abstract
The COP9 signalosome (CSN), an eight‐subunit protein complex, is conserved in all higher eukaryotes. CSN intersects the ubiquitin–proteasome pathway, modulating signaling pathways controlling various aspects of development. We are using Drosophila as a model system to elucidate the function of this important complex. Transcriptome data were generated for four csn mutants, sampled at three developmental time points. Our results are highly reproducible, being confirmed using two different experimental setups that entail different microarrays and different controls. Our results indicate that the CSN acts as a transcriptional repressor during development of Drosophila , resulting in achronic gene expression in the csn mutants. ‘Time shift’ analysis with the publicly available Drosophila transcriptome data indicates that genes repressed by the CSN are normally induced primarily during late embryogenesis or during metamorphosis. These temporal shifts are likely due to the roles of the CSN in regulating transcription factors. A null mutation in CSN subunit 4 and hypomorphic mutations in csn5 lead to more severe defects than seen in the csn5‐null mutants strain, suggesting that CSN5 carries only some of the CSN function. ### Synopsis The COP9 signalosome (CSN) is a highly conserved regulatory protein complex that in higher eukaryotes consists of eight subunits named CSN1 to CSN8 ([Wei and Deng, 2003][1]). The most studied CSN function is regulation of protein degradation. CSN interacts with E3‐ubiquitin ligases, removing Nedd8, a ubiquitin‐like modifier, from cullin‐based E3s ([Lyapina et al , 2001][2]), thereby regulating ligase activity. The CSN also mediates phosphorylation and deubiquitination of ubiquitin–proteasome pathway substrates, and as a consequence alters their stability and subcellular localization (reviewed in [Harari‐Steinberg and Chamovitz, 2004][3]). Loss‐of‐function mutants in Drosophila CSN4 and CSN5 are larval lethal and display both common and distinct phenotypes ([Oron et al , 2002][4]; [Harari‐Steinberg et al , 2007][5]). The lack of complete phenotypic overlap between the csn mutants can be explained by at least two non‐exclusive hypotheses. First, each subunit can have different roles within the CSN complex. Second, each subunit can have distinct roles independent of the CSN. We further hypothesized that there could be numerous CSN‐regulated pathways that are not morphologically evident. To clarify these issues, we initiated a global analysis of transcription profiles on our available CSN mutants. Two independent rounds of microarray analysis were carried out using two null alleles ( csn4 null and csn5 null ) ([Oron et al , 2002][4]) and two hypomorhic alleles ( csn5 1 and csn5 3 ) ([Suh et al , 2002][6]) in two CSN subunits (CSN4 and CSN5) examined at three developmental time points, 60, 72, and 96 h after egg deposition (AED). In both rounds of experimentation, the expression levels of ∼20% of the genes on the microarrays were found to change significantly. The two rounds gave highly similar results, with up to ∼90% overlap in identification of misregulated genes, showing a high robustness of the results. Hierarchical clustering analysis indicated two major trends. First, dendrogram clades center on developmental time points rather than individual mutants, indicating that different changes occur throughout development, but these changes are similar among the different mutants. Second, while fewer genes are misregulated in csn5 null relative to the other mutants, most of the expression profile of csn5 null is shared with csn4 null . This suggests that part of the molecular phenotype of csn4 null is due to the absence of CSN5. However, as CSN4 is essential for CSN complex integrity, while CSN5 is not ([Oron et al , 2002][4]), the additional genes misregulated in csn4 null could represent functions dependent on the entire CSN complex (which remains intact in csn5 null ) but not connected to CSN5‐mediated deneddylation. This has major implications for our understanding of CSN function. The most celebrated function of the CSN is as a deneddylase, centered in CSN5 ([Cope et al , 2002][7]). However, if this was the only or even most central role for the CSN, we would expect that the transcriptome phenotype of the csn5 null mutant would be very similar to that of the csn4 null mutant. As csn4 null affects more genes than csn5 null , we conclude that the entire CSN has additional functions that are not dependent on CSN5. At 60 h AED, there were more up‐ than downregulated genes ([Figure 3B][8]). At this time point, all four mutants are morphologically indistinguishable from each other and wt in terms of body size and behavior. We therefore reasoned that at this time point, the underlying molecular differences between the mutant and wt larvae represent more primary effects of the mutations. We further hypothesized that the prevalence of up‐ rather than downregulated genes in the mutants indicates that the Drosophila CSN is a general repressor of various developmental/temporal cues that induce gene expression. In absence of the CSN repressor activity, as in the mutants, these genes would be expressed achronically. If this hypothesis is true, then groups of genes that are upregulated (derepressed) in the mutants at 60 h AED should be normally induced in the wt at different developmental stages. To test our hypothesis, we used the available data sets from the developmental time‐course expression profiling of wt Drosophila ([Arbeitman et al , 2002][9]). We mined for the normal time of induction in the wt of genes that were derepressed in the csn mutants at 60 h AED. Three classes of genes are identified: genes normally upregulated before 60 h AED, genes that are normally upregulated after 60 h AED, and genes that are normally induced in both early and...