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

Efrat Oron¹, Tamir Tuller², Ling Li³, Nina Rozovsky¹, Daniel Yekutieli⁴, Sigal Rencus-Lazar⁵, Daniel Segal⁵, Benny Chor², Bruce A Edgar³ & Daniel A Chamovitz¹

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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). 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), 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).

Loss-of-function mutants in Drosophila CSN4 and CSN5 are larval lethal and display both common and distinct phenotypes (Oron et al, 2002; Harari-Steinberg et al, 2007). 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) and two hypomorhic alleles (csn5¹ and csn5³) (Suh et al, 2002) 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), 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). 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). 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.

Figure 3

(A) Morphology of csn mutants and wt at 60, 72, and 96 h AED. (B) Distribution of up- and downregulated genes in the mutants at the time points checked. The stars designate time points with more up- than downregulated genes. Only genes with >1.85-fold differential expression and FDR<0.05 were considered.

Full figure and legend (309K)Figures & Tables index

To test our hypothesis, we used the available data sets from the developmental time-course expression profiling of wt Drosophila (Arbeitman et al, 2002). 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 late development, relative to 60 h AED. We termed these genes 'time shifted'.

To determine if CSN-dependent repression of gene expression is time specific, we plotted the number of genes identified in each time point as a function of their normal time of induction (Figure 7). As a control, we sampled random groups of genes from the data set. Transcript levels of the time-shifted genes normally increase in the wt predominantly at one of two time windows—either during late embryogenesis (16–24 h) or late larval/early metamorphosis (96–126 h).

Figure 7

Time-shift analysis. For each mutant, the wt expression levels of putatively time-shifted genes identified in Figure 6A were compared relative to their expression levels at T₆₀ such that at T₆₀, the relative value=1 (base line). The y-axis shows the number of genes upregulated (e.g. relative value >1) at a particular time point. The black lines represent the average of >100 random gene groups that were sampled. Note that for csn5^null, the analysis was carried out using T₇₂. The total number of genes in each analysis is different as detailed in Figure 6A and is presented here as normalized.

Figure 6

Genes upregulated at 60 h AED in the mutants are putatively time shifted. (A) Percentage of upregulated genes that are putatively time shifted in the first (6k) and second (12k) rounds of microarray analysis, and their predicted normal time of induction. The number in each bar is absolute number of genes. (B) Snapshot of wt developmental time-course data (Based on data in Arbeitman et al, 2002) for genes putatively time shifted in the csn mutants. Representative time-shifted genes that were identified in both the first and second sets of experimentation are shown for each category (see Supplementary information for complete lists). Yellow shows upregulation while blue shows downregulation.

Full figure and legend (126K)Figures & Tables index

Full figure and legend (286K)Figures & Tables index

GO analysis revealed that >70% of the time-shifted genes are clearly involved in some aspect of development or signaling. Several of the significant GO groups identified are closely associated with cellular stress responses or molting.

A group of genes implicated in ecdysone signaling are misexpressed in both csn4 and csn5 mutants. While in csn4^null a majority of these genes show abnormal expression already at 60 h AED, in csn5^null the misexpression is evident only at 72 h AED. This raises the possibility that the reason that csn5^null does not display the molting phenotypes clearly observed in csn4^null is not because that CSN5 and CSN4 regulate different pathways, but rather because of different temporal disruption of the same pathways.

A major difficulty in analysis of mutants in the COP9 signalosome is determining which of the many phenotypes arise directly from the lesions in the CSN and which are pleiotropic downstream effects. That mutation in CSN leads to accumulating downstream effects is clear as the number of misregulated genes increases with time in all mutants, as does the overlap between the mutants.

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