Combinatorial mapping of E3 ubiquitin ligases to their target substrates

E3 ubiquitin ligases (E3s) confer specificity of protein degradation through the recognition of specific target substrates for ubiquitination and subsequent degradation, making E3s attractive candidates for drug development. There are over 600 annotated E3s in the human genome, yet the vast majority have no known substrate proteins, due in part to the limited scalability of methods to evaluate the vast number of potential E3-substrate interactions. To address this, we developed COMET (Combinatorial Mapping of E3 Targets), a combinatorial framework that can test the role of many specific E3s in regulating the abundance of many specific substrates, within the context of a single experiment. As a proof-of-concept, we applied COMET to identify specific E3 subunits of the SCF ubiquitin ligase complex that mediate the degradation of target substrates, including short-lived transcription factors (TFs). Our data suggest that many E3-substrate relationships are complex rather than simple 1:1 associations. Further, we provide computational models of E3-substrate interactions from our screen. Looking forward, we anticipate that COMET can be further scaled to enable comprehensive mapping of regulators of protein stability to their target substrates Overall design: Combinatorial plasmid libraries of E3 ubiquitin ligase gRNAs and potential E3 ubiquitin ligase substrates were cloned. These libraries were integrated into K562-Cas9-rtTA cells, which were then sorted into four FACS bins based on the GFP:mCherry ratio. Sequencing libraries were prepared from each bin with PCR replicates.