The Caenorhabditis elegans CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells.

BACKGROUND: Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified. RESULTS: We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling. CDT-2 acts redundantly with negative modulators of LET-23 signalling and CDT-2 or CUL-4 downregulation causes persistent expression of the egl-17::cfp transgene, a marker of LET-23 signalling during vulva development. In addition, we show that CDT-2 physically interacts with SEM-5 (GRB2), a known negative modulator of LET-23 signalling that directly binds LET-23, and provide genetic evidence consistent with CDT-2 functioning at or downstream of LET-23. Interestingly, both SEM-5 and CDT-2 were identified independently in a screen for genes involved in receptor-mediated endocytosis in oocytes, suggesting that attenuation of LET-23 by CDT-2 might be through regulation of endocytosis. CONCLUSIONS: In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells. In future, it will be interesting to investigate the potential link to endocytosis and to determine whether other signalling pathways dependent on endocytosis, e.g. LIN-12 (Notch) could be regulated by this ubiquitin ligase complex. This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation.

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression

A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity

Lineage specific trimethylation of H3 on lysine 4 during C. elegans early embryogenesis

AbstractIn many organisms early embryogenesis is characterised by a period refractory to transcription. In Caenorhabditis elegans, the one-cell embryo is transcriptionally inactive, but at around eight-cell stage transcription is activated in the somatic lineage. This model suggests that histone tail modifications associated with activation of transcription, such as di- or trimethylation of histone 3 on lysine 4 (H3K4me2/me3) should be enriched in the somatic lineage. Here, we have investigated the deposition of H3K4me3 during embryogenesis and found that it is more dynamic than anticipated. In the eight-cell stage embryo, H3K4me3 deposition is poor in the germline blastomere, as expected, but surprisingly three somatic blastomeres also remain poor in H3K4me3. All the other somatic blastomeres show robust deposition of H3K4me3. Interestingly, the three somatic blastomeres poor in H3K4me3 are descendants of the first germline blastomere, implying an activity that impedes on H3K4me3 deposition in these cells. In contrast, the deposition of H3K4me2 and H3K27me2/3 is not lineage restricted. Taken together, our data reveal that H3K4me3 deposition is highly regulated according to the cell lineage involved

An RNAi-based dimorphic genetic screen identified the double bromodomain protein BET-1 as a sumo-dependent attenuator of RAS-mediated signalling

Attenuation of RAS/RAF/MAPK signalling is essential to prevent hyperactivation of this oncogenic pathway. In C. elegans, the sumoylation pathway and a combination of histone tail modifications regulate gene expression to attenuate the LET-60 (RAS) signalling pathway. We hypothesised that a number of chromatin regulators are likely to depend on sumoylation to attenuate the pathway. To reveal these, we designed an RNAi-based dimorphic genetic screen that selects candidates based on their ability to act as enhancers of a sumo mutant phenotype, such interactions would suggest that the candidates may be physically associated with sumoylation. We found 16 enhancers, one of which BET-1, is a conserved double bromodomain containing protein. We further characterised BET-1 and showed that it can physically associate with SMO-1 and UBC-9, and that it can be sumoylated in vitro within the second bromodomain at lysine 252. Previous work has shown that BET-1 can bind acetyl-lysines on histone tails to influence gene expression. In conclusion, our screening approach has identified BET-1 as a Sumo-dependent attenuator of LET-60-mediated signalling and our characterisation suggests that BET-1 can be sumoylated

Comparative analysis between the double <i>smo-1</i>_<i>lf</i> bet<i>-</i>1_<i>lf</i> mutant and the respective single mutants.

<p>A) Two alternative models (linear and parallel) for attenuation of the LET-60(RAS) signalling pathwayby SMO-1 and BET-1. B) The percentage of VPCs expressing <i>egl-17::cfp</i> is indicated on the y axis. Each bar represents the descendants of the indicated parental VPC. The genotype analysed is indicated in the inset legend. We analysed a minimum of 40 animals for each genotype. B) Western blots performed in triplicate, and prepared from larval stage 4 (L4) against phosphorylated MPK-1 (top panel), MPK-1 (middle panel) and tubulin (bottom panel). Quantification of pMPK-1 is relative to tubulin levels. </p