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.

Automated summary

All multicellular organisms rely on differential gene expression, which is regulated by regulatory DNA elements and DNA-binding transcription factors. This study focuses on the less-studied repressors and develops a high-throughput method called repressive-domain (RD)-seq to systematically identify repressive domains (RDs) in complex DNA-fragment libraries. By screening more than 200,000 fragments in Drosophila melanogaster, the researchers identify 195 RDs in known repressors and proteins not previously associated with repression. They find that many RDs contain recurrent short peptide motifs that are conserved between fly and human and are functionally important. Moreover, they show that different RDs interact with and depend on different co-repressors (CoRs). These findings provide valuable insights into the sequences and functional impact of repressors and should benefit further studies.