This strategy identified CDT 2, an evolutionary conserved homologue of human CDT2 also called DTL or DCAF2. Human CDT2 was first discovered as a transcript down regulated following retinoic acid induced neuronal differentiation in pluripotent NT2 cells, suggesting a role in maintenance of self renewal capacity. CDT2, a WD40 sellckchem domain containing protein, was later found associated to the CUL4 DDB1 E3 ubiquitin ligase com plex. Within this complex, CDT2 acts as a sub strate recognition subunit. Two substrates have been well characterised, the license to replicate, CDT1, and the CDK inhibitor, p21. Degradation of CDT1 and p21 are essential to prevent rereplication or firing of origin of replication following DNA damage induced stress.
Therefore, CDT2, as part of the CUL4 DDB1 E3 ubiquitin ligase complex, plays a critical role in regula tion of DNA replication. Mouse CDT2 activity is essential for viability, which has precluded the study of its role during development. RNAi in C. elegans can sometime create knock down conditions that allow the identification of novel late onset activities. Here, we show that C. elegans CDT 2 and CUL 4 attenuate LET 23 signalling during vulva development. We found that SEM 5 phy sically interacts with CDT 2 and genetic studies are con sistent with CDT 2 acting at the level of the LET 23 receptor. Finally, we confirmed that CDT 2 and SEM 5 are required for receptor mediated endocytosis in oocytes. We propose a model by which the CUL 4 DDB 1 CDT 2 ubiquitin ligase complex associates with SEM 5 to target LET 23 and regulate its endocytosis.
Methods Strains and general maintenance Strains were maintained as described in Brenner, lin 15AB is a temperature sensitive allele, which produces a lower penetrance Muv phenotype at 15 C than at 25 C. Strains genotypes are, gap 1, gap 1, lin 3, let 23, let 60, lin 15A, lin 15B, dpy 23, sem 5, sli 1, unc 101, cul 4 mIn1, unc 4 Dacomitinib II, arIs92, unc 119,pwIs23 unc 119, pwIs116. RNAi procedure Briefly, worms for RNAi exposure were synchronised using standard bleaching to isolate embryos. These were grown to the L3 stage and then transferred to RNAi plates. The mothers were transferred onto a new plate after three days and the F1 s laid on this plate were ana lysed, as previously described. For lin 3rf, let 23rf, and lin 45rf, F2 s were ana lysed instead of F1 s since the Vul phenotype leads to small broods.
RNAi clones used in this study were all confirmed by sequencing. Scoring of the Multivulvae phenotype Induction of vulval cells was scored by lineage analysis of vulval precursor cells. Briefly, L4 animals were mounted on agarose selleck chem pads and the descendants of the six Vulval Precursors Cells analysed to assign either the vulval fate or the non vulval fate. Each fully induced VPC is given a score of 1, a wild type vulva therefore has a score of three because three VPCs adopt the vulval fate. A score of 0.