A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [repressed STARR-seq]

Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a novel class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a novel highly-conserved domain ZAC and the corepressor G9a, independently of G9a's H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes. Overall design: Control and repressed focused STARR-seq experiments, using either a standard DSCP and RpS12 core promoter BAC-scale STARR-seq library mix or an equivalent mix with an invariant DLM3 silencer in the backbone, were perfomed in Drosophila melanogaster Schneider S2 cells. For both conditions two biological replicates were sequenced in paired-end mode, using 1% Drosophila pseudoobscura BAC-scale STARR-seq libraries as spike-in for sample normalization.