Identification and functional characterization of transcriptional activators in human cells

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale. [Display omitted] •Large-scale pooled tethering assay identifies ∼250 transcriptional activators in humans•Fragment screen pinpoints novel activation domains among screen hits•Proteomics, chemical inhibitors, and AlphaFold connect activators to their cofactors•Functional characterization of SRF-C3orf62, an oncogenic fusion found in myofibroma Alerasool et al. use a pooled assay to identify over 200 transcriptional activators in the human proteome. They also employ fragment screens to identify short activation domains among the hits, connect activators by proximity biotinylation and AlphaFold predictions to distinct co-activators, and functionally characterize several previously unknown transcriptional activators.