High-Throughput Discovery and Characterization of Human Transcriptional Effectors

Josh Tycko, Nicole DelRosso, Gaelen T. Hess, Aradhana, Abhimanyu Banerjee, Aditya Mukund, Mike V. Van, Braeden K. Ego, David Yao, Kaitlyn Spees, Peter Suzuki, Georgi K. Marinov, Anshul Kundaje, Michael C. Bassik*, Lacramioara Bintu*

Thousands of proteins localize to the nucleus, however it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit - a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several Domains of Unknown Function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find new repressors as short as 10 amino acids. Finally, we report new activator domains, including a divergent KRAB. Together, these results provide a resource of 600 human proteins containing effectors, and demonstrate a scalable strategy for assigning functions to protein domains.