Description:

Traditional functional proteomics approaches, such as interaction proteomics, have generally relied on the guilt-by-association principle. That is, function of novel proteins is inferred from their association patterns with proteins with a known function. While this approach has worked remarkably well and still forms the foundation of most omics-based discovery studies, it is often challenging and time-consuming to characterize the function of the protein even if the association is known. To address this challenge, we have established a screening platform to discover protein function with activity-based readouts. Here, we force proteins to interact with each other with chemically inducible dimerization systems or other targeting methods. One protein (sensor) provides a readout for the biological function, and the other (potential effector) is assayed for this function. In one application, we tethered the human ORFeome to specific genomic loci with dCas9 and identified hundreds of proteins that regulate transcriptional activation or repression – many of which are previously uncharacterized proteins. We identified activation domains in these novel transcriptional activators with a systematic tiling approach and connected them to specific co-activator complexes with proximity biotinylation coupled to mass spectrometry. In a similar manner, we have identified repressors that repress transcription more potently than currently used CRISPRi screening platforms. These results illustrate the potential of functional tethering screens to identify novel regulators of biological processes.

Mikko Taipale, PhD
Research Chair in Functional Proteomics and Proteostasis; CIFAR Azrieli Global Scholar
Assistant Professor, Cellular and Biomolecular Research
University of Toronto