The molecular and genetic analysis of C. elegans cullins CUL-2 and CUL-4

Abstract

Members of the cullin family function as integral components of cullin/RING finger ubiquitin ligase complexes.Here we demonstrate that two C.elegans cullins, CUL-2 and CUL-4,are key cell cycle regulators.We discovered that CUL-2 promotes the G1-to-S phase transition by negatively regulating CKI-1,a member of the CIP/KIP family of cyclin-dependent kinase inhibitors.CUL-2 is also required for mitotic chromosome condensation.In cul-2 mutant embryos,mitotic chromosomes fail to condense,leading to multiple nuclei,DNA bridges,and unequal DNA segregation.To explore the composition of CUL-2/RING finger complexes,potential C.elegans CUL-2 interactors were uncovered.By BLAST search and RNAi,all C.elegans orthologs of human CUL2/VCB components but VHL were suggested to function with CUL-2.In addition,over ten proteins that associate with FLAG-tagged CUL-2 were identified by affinity purification and mass spectrometry.The identification of four BC-box proteins as CUL-2 interactors suggests that CUL-2 forms multiple CUL-2/RING finger complexes, using Elongin BC to link BC-box proteins to the core complex.Interestingly,three transcriptional regulators were identified as CUL-2 interactors,suggesting a role for CUL-2 in transcription control.Another C.elegans cullin family member,CUL-4,is essential for maintaining genome stability by restraining DNA replication licensing in S phase.Inactivation of CUL-4 leads to an S phase arrest and massive DNA re-replication. To explore the mechanism of CUL-4 function,we investigated the regulation of the replication licensing factor CDT-1.We demonstrated that the C.elegans CDT-1 ortholog is required for DNA replication.Further,C.elegans CDT-1 is present in G1 phase nuclei but disappears as cells enter S phase.The absence of CDT-1 in S phase cells prevents DNA from undergoing re-replication.In cells lacking CUL-4,CDT-1 levels fail to decrease as cells enter S phase but instead remain constant in the re-replicating cells. Removal of one genomic copy of the cdt-1 gene suppresses the cul-4(RNAi )re- replication phenotype,suggesting that the aberrant presence of CDT-1 protein in S phase cells promotes re-replication.We propose that CUL-4 functions to prevent aberrant re- initiation of DNA replication,at least in part,by facilitating the degradation of CDT-1.