PT - JOURNAL ARTICLE AU - Nicole DelRosso AU - Josh Tycko AU - Peter Suzuki AU - Cecelia Andrews AU - Aradhana AU - Adi Mukund AU - Ivan Liongson AU - Connor Ludwig AU - Kaitlyn Spees AU - Polly Fordyce AU - Michael C. Bassik AU - Lacramioara Bintu TI - Large-scale mapping and systematic mutagenesis of human transcriptional effector domains AID - 10.1101/2022.08.26.505496 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.08.26.505496 4099 - http://biorxiv.org/content/early/2022/08/26/2022.08.26.505496.short 4100 - http://biorxiv.org/content/early/2022/08/26/2022.08.26.505496.full AB - Human gene expression is regulated by over two thousand transcription factors and chromatin regulators1,2. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of transcriptional effector domains they contain, their location in the protein, their activation and repression strengths, and the amino acids that are necessary for their functions. Here, we systematically measure the transcriptional effector activity of >100,000 protein fragments (each 80 amino acids long) tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 307 new activation domains and 592 new repression domains, a ∼5-fold increase over the number of previously annotated effectors3,4. Complementary rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine, and/or glutamine residues are necessary for activation domain activity. Additionally, the majority of repression domain sequences contain either sites for SUMOylation, short interaction motifs for recruiting co-repressors, or are structured binding domains for recruiting other repressive proteins. Surprisingly, we discover bifunctional domains that can both activate and repress and can dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of transcriptional effector domains provides a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression, and refining predictive computational models of effector domain function.Competing Interest StatementStanford has filed a provisional patent related to this work.