1. High-Throughput Discovery and Characterization of Human Transcriptional Effectors
- Author
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Abhimanyu Banerjee, Kaitlyn Spees, Nicole DelRosso, Georgi K. Marinov, Aradhana, Anshul Kundaje, David Yao, Josh Tycko, Michael C. Bassik, Gaelen T. Hess, Braeden K. Ego, Aditya Mukund, Lacramioara Bintu, Mike V. Van, and Peter Suzuki
- Subjects
Transcription, Genetic ,Protein domain ,Repressor ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Genes, Reporter ,Gene silencing ,Humans ,Amino Acid Sequence ,Gene Silencing ,Hox gene ,Promoter Regions, Genetic ,030304 developmental biology ,chemistry.chemical_classification ,Homeodomain Proteins ,0303 health sciences ,Effector ,Activator (genetics) ,Lentivirus ,Nuclear Proteins ,Reproducibility of Results ,Molecular Sequence Annotation ,Zinc Fingers ,Amino acid ,High-Throughput Screening Assays ,Repressor Proteins ,HEK293 Cells ,chemistry ,Mutation ,Homeobox ,Female ,Domain of unknown function ,CRISPR-Cas Systems ,K562 Cells ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
SummaryThousands 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 withHoxgenetic 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 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.
- Published
- 2020