Systematic identification of transcriptional activator domains from non-transcription factor proteins in plants and yeast.

Unlabelled: Transcription factors promote gene expression via trans-regulatory activation domains. Although whole genome scale screens in model organisms (e.g. human, yeast, fly) have helped identify activation domains from transcription factors, such screens have been less extensively used to explore the occurrence of activation domains in non-transcription factor proteins, such as transcriptional coactivators, chromatin regulators and some cytosolic proteins, leaving a blind spot on what role activation domains in these proteins could play in regulating transcription. We utilized the activation domain predictor PADDLE to mine the entire proteomes of two model eukaryotes, and ( ). We characterized 18,000 fragments covering predicted activation domains from >800 non-transcription factor genes in both species, and experimentally validated that 89% of proteins contained fragments capable of activating transcription in yeast. Peptides with similar sequence composition show a broad range of activities, which is explained by the arrangement of key amino acids. We also annotated hundreds of nuclear proteins with activation domains as putative coactivators; many of which have never been ascribed any function in plants. Furthermore, our library contains >250 non-nuclear proteins containing peptides with activation domain function across both eukaryotic lineages, suggesting that there are unknown biological roles of these peptides beyond transcription. Finally, we identify and validate short, 'universal' eukaryotic activation domains that activate transcription in both yeast and plants with comparable or stronger performance to state-of-the-art activation domains. Overall, our dual host screen provides a blueprint on how to systematically discover novel genetic parts for synthetic biology that function across a wide diversity of eukaryotes.

Significance Statement: Activation domains promote transcription and play a critical role in regulating gene expression. Although the mapping of activation domains from transcription factors has been carried out in previous genome-wide screens, their occurrence in non-transcription factors has been less explored. We utilize an activation domain predictor to mine the entire proteomes of and for new activation domains on non-transcription factor proteins. We validate peptides derived from >750 non-transcription factor proteins capable of activating transcription, discovering many potentially new coactivators in plants. Importantly, we identify novel genetic parts that can function across both species, representing unique synthetic biology tools.