Reporting in Molecular Cell, Marc Vidal and colleagues show, for the first time, how combining two established techniques — double-genetic-perturbation genetic analysis and large-scale protein interaction (interactome) mapping strategies, which identify functional and physical interactions, respectively — can help us to understand biological processes at a systems level in a metazoan organism.

The authors wanted to identify components of the human TGFβ signalling pathway, to try and better understand TGFβ signalling and its dysregulation in disease. As many of the known TGFβ-signalling-pathway components are highly conserved, they chose to study the Caenorhabditis elegans DAF7/TGFβ pathway.

First, Vidal and co-workers carried out a large-scale yeast two-hybrid (Y2H) screen using 6 known components of the DAF7/TGFβ pathway as bait. Of the 28 potential interacting proteins that were identified, 19 were used as bait in a second Y2H screen. In total, 71 interactions and 59 potential interacting proteins were identified, and over half of these proteins have strong sequence similarity to human proteins. The physical interactions of the interactome map were then validated using co-affinity purification assays.

To find out about the functional relationships in this pathway, Vidal and colleagues used RNA interference (RNAi) to knock down genes that were derived from the interactome map in various loss-of-function mutant backgrounds that correspond to known DAF7/TGFβ-pathway components. This double genetic perturbation identified 13 genetic interactions among 5 of the known DAF7/TGFβ-pathway genes and 9 of the genes that were derived from the interactome map.

The authors went on to characterize one of these genes (W01G7.1) and identified it as daf-5 . They suggest that W01G7.1/DAF5 is the C. elegans homologue of human SNO/SKI, which functions in the mammalian TGFβ pathway. The remaining 8 proteins are not known to have a role in TGFβ signalling in any species.

As both RNAi and Y2H have their shortcomings, and generate a number of false-negative results, the authors caution that there are likely to be many more genes and interactions involved in the DAF7/TGFβ pathway than were identified by this study. Nevertheless, this study does reveal that the DAF7/TGFβ pathway is more complex than present models indicate.

The future of this combined approach looks promising. As the C. elegans genome-scale gene-knockout project progresses and more genetic mutants become available, more in-depth functional analyses could be possible and the power of this combined approach should only increase.