By Liangyu Zhang and Abby F. Dernburg
The nematode Caenorhabidis elegans is among the most widely used and powerful model organisms for studying mechanisms underlying cellular and developmental processes. Although a variety of approaches for conditional protein expression have been developed in C. elegans, available tools for conditional protein depletion are far more limited, particularly in the germ line. We were thus motivated to develop a technique to control the abundance of proteins in living animals, which we felt would be a great addition to the toolbox available in this system.
In the Dernburg lab, we are interested in studying the molecular mechanisms underlying meiosis. In many cases we can use genetic mutations to interrogate the roles of individual proteins, but this approach does not work well if the proteins perform essential functions during mitosis, which is required for proliferation of the germ line. We therefore sought to develop a method that would enable inducible, rapid, and quantitative protein depletion in the germ line. After considering a number of possible strategies, we focused on the auxin-inducible degradation (AID) system, which has been applied in cultured cells and single-celled organisms (Nishimura et al., 2009). This approach was originally adapted from the plant auxin perception system. Inducible degradation relies on a small molecule phytohormone produced by all plants, auxin (indole-3-acetic acid). In addition, it requires TIR1, an F-box protein, which forms part of an Skp1–Cullin–F-box (SCF) E3 ubiquitin ligase complex. In the presence of auxin, TIR1 recognizes peptide sequences (degrons) that are present in a large number of target proteins, mostly transcriptional regulators, expressed by plants, and targets these proteins for polyubiquitylation and proteasome-mediated degradation. We thought this system might be transplantable to nematodes, since auxin is a very small, fairly water-soluble molecule, potentially making it easy to deliver to living animals and readily diffusible through tissues.
A rotation student, Ze Cheng, first accepted the challenge of adapting the AID system to C. elegans. He generated a number of constructs to test its feasibility. Because there is no endogenous auxin receptor in C. elegans, he made a strain stably expressing a modified Arabidopsis thaliana TIR1 from a single copy transgene using MosSCI; (Frokjaer-Jensen et al., 2008). He also made a construct to express a degron- and GFP-tagged SMU-2 from extrachromosomal arrays in the germ line. After treating worms expressing both constructs with auxin for a couple of hours, he was excited to observe a disappearance of the green fluorescent SMU-2::GFP signal, indicating the AID system might indeed be useful in C. elegans.
We then systematically expanded and tested the AID system in C. elegans. To maximize the chances of success, we incorporated two amino acid changes in AtTIR1 that were found to increase its binding affinity for degron-tagged substrates and to thereby increase its sensitivity to auxin (Yu et al., 2013). We created strains expressing TIR1 under control of various promoters and 3’ UTR sequences to drive germ line-specific or temporally regulated expression. We used a 44-amino acid minimal degron sequence derived from Arabidopsis thaliana IAA17 (Morawska and Ulrich, 2013) and tagged proteins of interest with this sequence using CRISPR/Cas9-mediated editing (Dickinson et al., 2013). Gratifyingly, we detected efficient inducible-degradation in the germ line when growing the transgenic worms with auxin-containing liquid culture or plates. Excitingly, rapid degradation was consistently achieved within a reasonable range of auxin concentrations (<1 mM), which did not seem to have any effects on wild-type worms. We also found that the position of the degron was quite flexible; it could be placed at either end of a target protein, or even sandwiched between the target and another tag, such as GFP.
Considering the potential usefulness of the AID system for the research community, we further analyzed the inducible degradation of target proteins in the soma of C. elegans in detail. To do this, we made a variety of tissue-specific TIR1 strains with tissue-specific promoters and 3’ UTR sequences. We then combined these TIR1 transgenes with different types of degron-tagged transgenic targets. We found either cytoplasmic or nuclear proteins tagged with the degron can be tissue specifically degraded at various developmental stages in an auxin concentration dependent manner. After trying a wide range of auxin, we also noticed that the degradation is reversible upon auxin removal, with lower auxin doses accelerating recovery. To our surprise, we further detected efficient inducible degradation of targets in early embryos inside the mother and in laid eggs, suggesting promising usefulness of the AID system for studying mechanisms underlying embryo development. Notably, the inducible degradation is also efficient in the absence of food, making it useful for studying starvation-induced processes, such as autophagy and larvae arrest.
When we shared these findings with some colleagues, they were extremely excited about the potential of the system. One of them, Jordan Ward at UCSF, wanted to test the ability of this system to address key questions underlying nuclear hormone receptor-mediated control of developmental gene regulatory networks. He was able to tag two essential nuclear hormone receptors, NHR-23 and NHR-25, with the degron sequence and found that each could be depleted within 40 min, enabling detailed functional dissection of these proteins during development.
Another exciting finding was that the AID system can produce more penetrant phenotypes than depletion by RNAi, not only in the worm soma but also in the germ line. Given the high efficiency of CRISPR/Cas9-mediated genome editing in C. elegans, tagging proteins of interest with the 44-amino acid degron is now quite easy and fast, further augmenting the utility of the AID system for cell and developmental studies in worms.
We have made the AID-related worm strains and plasmids available through the CGC and Addgene, respectively. A large number of worm laboratories have already tried the AID system to deplete proteins of interest in C. elegans. We are getting great feedback from our colleagues, several of whom have told us that the AID system works spectacularly in their hands. In principle, this approach may be applicable to a wide range of other organisms. We thus look forward to seeing the application of this technology in not only worm labs but also other metazoan model organism labs.
Full article at: http://dev.biologists.org/content/142/24/4374.long
Dickinson, D. J., Ward, J. D., Reiner, D. J. and Goldstein, B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028-1034.
Frokjaer-Jensen, C., Davis, M. W., Hopkins, C. E., Newman, B. J., Thummel, J. M., Olesen, S. P., Grunnet, M. and Jorgensen, E. M. (2008). Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375-1383.
Morawska, M. and Ulrich, H. D. (2013). An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30, 341-351.
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. and Kanemaki, M. (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917-922.
Yu, H., Moss, B. L., Jang, S. S., Prigge, M., Klavins, E., Nemhauser, J. L. and Estelle, M. (2013). Mutations in the TIR1 auxin receptor that increase affinity for auxin/indole-3-acetic acid proteins result in auxin hypersensitivity. Plant Physiol. 162, 295-303.