The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability

Abstract

The COP9 signalosome (CSN) is required for the full activity of cullin-RING E3 ubiquitin ligases (CRLs) in eukaryotes. CSN exerts its function on CRLs by removing the ubiquitin-related NEDD8 conjugate from the cullin subunit of CRLs. CSN seems, thereby, to control CRL disassembly or CRL subunit stability. In Arabidopsis thaliana, loss of CSN function leads to constitutive photomorphogenic (cop) seedling development and a post-germination growth arrest. The underlying molecular cause of this growth arrest is currently unknown. Here, we show that Arabidopsis csn mutants are delayed in G2 phase progression. This cell cycle arrest correlates with the induction of the DNA damage response pathway and is suggestive of the activation of a DNA damage checkpoint. In support of this hypothesis, we detected gene conversion events in csn mutants that are indicative of DNA doublestrand breaks. DNA damage is also apparent in mutants of the NEDD8 conjugation pathway and in mutants of the E3 ligase subunits CULLIN4, COP1 and DET1, which share phenotypes with csn mutants. In summary, our data suggest that Arabidopsis csn mutants undergo DNA damage, which might be the cause of the delay in G2 cell cycle progression.KEY WORDS: COP9 signalosome, Cell cycle, DNA damage Development 135, 2013Development 135, -2022Development 135, (2008 (Miséra et al., 1994; Chory et al., 1989;Deng et al., 1991). COP1 is a RING-type E3 ubiquitin ligase that mediates the degradation of several positive photomorphogenesis regulators (Osterlund et al., 2000;Seo et al., 2003;Seo et al., 2004). The human COP1 ortholog (RFWD2) has been implicated in c-JUN degradation and in DNA damage response following irradiation Wertz et al., 2004). Human COP1 is inactivated in response to DNA damage by ATM-dependent phosphorylation, and the consequent stabilization of its degradation target p53 induces a G1 cell cycle arrest . The function of DET1 only became clear when it was recognized that it is a subunit of a CULLIN4 (CUL4)-containing CRL, designated DCX DET1COP1 or CUL4-DDB1 DET1COP1 , which also includes COP1 and the adaptor subunit DAMAGED DNA-BINDING PROTEIN1 (DDB1) (Benvenuto et al., 2002;Dornan et al., 2004;Wertz et al., 2004;Yanagawa et al., 2004;Chen et al., 2006). Although these findings suggest that COP1 and DET1 function together in a CUL4-containing CRL, the COP1 monomer alone has in vitro E3 ligase activity. It is therefore presently unclear which functions of COP1 require the E3 complex (and DET1) and which functions are mediated by COP1 alone.Arabidopsis csn mutants arrest growth at the seedling stage. The underlying molecular cause of this growth arrest remains to be identified. Here we show that csn mutant cells have a delay in G2 phase progression. This delay correlates with the activation of the DNA damage response pathway but is not exclusively induced by the DNA damage signaling kinases ATAXIA TELANGIECTASIA MUTATED (ATM) or WEE1. Our observation that gene conversion events can occur in csn mutants strongly argues that DNA doublestrand break...

Auxin Responses in Mutants of the Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC9 Signalosome

“…To examine whether the three csn mutants differ from each other at the gene expression level, we examined a set of gene expression data that had been obtained from 7-d-old dark-and light-grown wildtype, csn3-1, csn4-1, and csn5ab mutant seedlings using Affymetrix ATH1 arrays (Dohmann et al, 2008). These microarray data were analyzed using the R statistical programming environment and the Bioconductor modules Limma and Affy (Ihaka andGentleman, 1996, 2004;Irizarry et al, 2002;Smyth, 2004).…”

“…The material was processed and hybridized to Affymetrix ATH1 gene chips as described (Dohmann et al, 2008). The microarray data can be retrieved as experiment GSE9728 at GeneExpressionOmnibus (GEO).…”

The GATA-type transcription factors GNC and GNL/CGA1 repress gibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS

“…To generate GNC Pro :GUS and GNL Pro :GUS, 2-kb promoter fragments were amplified from Col-0 genomic DNA using GNC Pro-LP/RP and GNL Pro-LP/RP, respectively, and inserted as EcoRI-BglII fragments into pCAM-BIA1391Z. At least six transgenic lines were generated in the Col-0 ecotype and analyzed at the histochemical level as described previously (Dohmann et al 2008). For GUS staining, seeds were fixed in 20% acetone for >12 h at À20°C prior to staining.…”

Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

“…Other functions of the CSN complex identified in mammalian cells include regulating the phosphorylation of ubiquitin-proteasome pathway substrates through CSN-associated kinases (7,(15)(16)(17)(18). Overall, the CSN complex appears to be a key player in protein subcellular localization (19,20), DNA-damage response (21), NF-κB activation (22), development, and cell cycle control (23,24). Thus, the functions of the CSN complex are beyond the regulation of light-dependent reaction in plants.…”