Systematic identification and characterization of repressive domains in Drosophila transcription factors.

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA‐binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co‐repressors (CoRs) they recruit have not been determined. Here, we develop a high‐throughput, next‐generation sequencing‐based screening method, repressive‐domain (RD)‐seq, to systematically identify RDs in complex DNA‐fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription‐related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequence‐altering mutations and should provide a valuable resource for further studies. Synopsis: Transcriptional repressors and their respective repressive domains (RD) have, in contrast to activators, not been systematically characterized. Here, high‐throughput identification of RDs via a newly developed RD‐seq screening approach reveals that RDs contain short recurrent and conserved peptide motifs required for the recruitment of co‐repressors and repressive function. RD‐seq identifies known and novel transcriptionally repressive domains.RDs contain short peptide motifs that are required for the repressive function.Repressive peptide motifs can predict the interacting co‐repressor.RD function and repressive motifs are conserved throughout evolution. [ABSTRACT FROM AUTHOR]