A high-throughput method to identify trans-activation domains within transcription factor sequences.

Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans-activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high-throughput approach tAD-seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix-loop-helix domains that are typically annotated as DNA-binding. We also identified tADs in the non-native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which-particularly for TFs of different transcription activating functionalities-is still poorly understood.