From stem cells to gametes : CUL-2-mediated protein degradation in the formation and maintenance of the C. elegans germline – CRL2INGSC

C. elegans is a multicellular organism with a simple anatomy and an invariant cell lineage, ideally suited to study the role of the ubiquitin-proteolytic system during development using a combination of genetics and cell biology approaches. However, proteomic approaches that are powerful to study the ubiquitin-proteolytic system are more difficult to use in this system for several reasons. First, there is currently no available method to purify ubiquitinated proteins in C. elegans. Second, the material is not always easy to obtain in sufficient quantities for biochemical approaches. Whereas C. elegans has been instrumental for the identification of enzymes of the ubiquitin-proteolytic system (e.g cullins) through genetic approaches, not a single ubiquitination site has been mapped on a C. elegans protein so far. The emergence of new methods allowing genome editing (CRISPR/Cas9) in C. elegans, stresses the importance of identifying targets of the ubiquitin-proteolytic system, and particularly their ubiquitination site, to understand the role of the system during development of multicellular organisms. For all these reasons we have set up proteomic approaches in C. elegans. First, using the CRISPR/Cas9 system, we have generated a C. elegans line expressing ubiquitin in fusion with a 6xHis tag specifically in the germline and in the early embryo allowing the purificatin of ubiquitinated proteins in denaturing conditions, which preserve ubiquitin on target proteins. This is a major technical advance because purifying and identifying ubiquitinated proteins using mass spectrometry approaches was clearly a bottleneck to the study of the ubiquitin-proteolytic system in C. elegans. Second, we have systematically identified ubiquitination sites (GlyGlyome) on C. elegans proteins through proteomic approaches.

We have identified during the course of this project a novel CUL-2 target called SPAT-1. SPAT-1 is essential for the activation of the Polo-Like kinase PLK-1 and for the development of the germline and mitotic entry in the early embryo. We have studied SPAT-1 regulation and showed that SPAT-1 multisite phosphorylation by the CyclinB/Cdk1 kinase promotes SPAT-1/PLK-1 interaction and triggers PLK-1 phosphorylation on its activating T-loop by the Aurora A kinase. Once activated, PLK-1 promotes mitotic entry in the early embryo. Importantly, this regulatory pathway leading to PLK-1 activation is conserved between worms and humans. Indeed, we have shown that Cdk1/CyclinB promotes Plk1 activation through Bora (SPAT-1 homologue in humans) phosphorylation in human cells. A manuscript presenting these data has been reviewed favourably in The Journal Of Cell Biology (currently in revision).
In order to identify CUL-2 targets through global approaches we have generated a cell line expressing ubiquitin in fusion with the 6xHis tag allowing the purification of ubiquitinated proteins in denaturing conditions. This line will be useful for the community because as mentioned previously this reagent has not been generated in C. elegans. In parallel, we have identified around 8000 ubiquitination sites in collaboration with cell signalling. In summary, during the first phase of the project, we have identified a novel CUL-2 target and developed tools to identify CUL-2 targets through global proteomic approaches. These methods will complement the genetic approaches.

Our work aimed at identifying novel targets of the ubiquitin-proteolytic system and at elucidating the functional role of their degradation, in space and time, during the development of the germline and the early embryo has several implications. First, as mentioned earlier, no strategy has been envisioned yet to identify targets of the ubiquitin-proteolytic system through global proteomic approaches in C. elegans. Therefore, we anticipate that several studies will be initiated following our release of the ubiquitination sites on C. elegans proteins. Second, we anticipate that most of our findings in C. elegans will be transposable in other systems and possibly in humans. For instance, we have shown that the regulation of SPAT-1/Bora is conserved between worms and humans.

- Identification of a role of the CRL2 E3-ligase in the establishment and maintenance of the C. elegans germline (Burger et al. PLoS Genetics 2013)
- Identification of SPAT-1 as a novel CUL-2 target and study of its regulation (Tavernier et al. The Journal Of Cell Biology, in revision)
- Generation of a C. elegans line expressing Ubiquitin in fusion with the 6xHis tag (in preparation)
- Systematic identification of ubiquitination sites in C. elegans proteins (in preparation)

Precise control of the transition from self-renewal to terminal differentiation in stem cells is critical to maintain a balance between cell populations: an excess of stem cell self-renewal can lead to tumourigenesis, whereas an excess of differentiation can deplete the stem-cell pool. In the adult Caenorhabditis elegans germline, Notch signals emanate from the somatic distal tip cell to maintain germline stem cells (GSCs) in a proliferative state by repressing the translation of meiotic promoting factors. We have uncovered a novel pathway regulating the decision between GSC renewal and meiotic differentiation that involves the ubiquitin-proteolytic system (UPS). Using a novel temperature-sensitive allele of the cul-2 gene, we found that the CUL-2 RING E3 ubiquitin ligase, in combination with the Leucine Rich Repeat 1 substrate recognition subunit (CRL2LRR-1), negatively regulates the transition from the mitotic zone of the germline to the meiotic programme of chromosome pairing, synapsis, and recombination. More specifically, we find that CRL2LRR-1 regulates in stem cells the stability of the HORMA domain-containing protein HTP-3, which is required for loading structural proteins onto meiotic chromosomes and for the formation of the double-strand breaks that initiate meiotic recombination. Furthermore, we found that cyclin E/Cdk2 kinase, which is specifically activated in GSCs but repressed upon meiotic differentiation, phosphorylates HTP-3 and regulates its stability. Besides HTP-3, CUL-2 targets other factors for degradation to prevent precocious meiotic entry and to promote germline stem cell proliferation. Herein, we propose to use a unique combination of genetics, cell biology, biochemical and quantitative proteomic approaches to elucidate the role of protein degradation in germ cell biology. In particular, we propose to identify CUL-2 targets in the germline and the molecular mechanisms controlling their degradation in space. The role of the UPS and CUL-2 in germline stem cell biology has not been studied so far. CUL-2 is evolutionarily conserved in metazoans and appears to regulate germ cell divisions in Drosophila. Therefore emerging paradigms provided by the study of germ cell biology in C. elegans should be directly applicable in other systems and possibly also in humans.